Hydrogel preparation and process of manufacture thereof

ABSTRACT

A polymeric hydrogel having a network of a macropores and micropores formed by copolymerizing at least one monomer having at least one double bond and at least one crosslinker having at least two double bonds in the presence of an organic additive forming a hydro-organic system with water, and uses thereof as separation media.

TECHNICAL FIELD

The present invention relates to a separation medium comprising ahydrogel preparation consisting of macropores and micropores obtainableby using a hydro-organic solvent.

BACKGROUND ART

Hydrogels for Separation Processes

In many applications of separation processes, it is desirable to have aporous matrix with good water compatibility and mechanical properties.In general, two broad classes of matrixes have been used. One generalclass is derived from water insoluble polymers by precipitationprocedures such as Diffusion Induced Phase Separation (DIPS) andThermally Induced Phase Separation (TIPS). These matrixes are relativelyhydrophobic. A typical example is polysulphones membranes, whichsometime require surface treatment or modification by physicaladsorption of hydrophilic polymers (e.g. poly(vinyl alcohol)) to achievesatisfactory water wetting properties.

In many applications it is preferred to synthesize hydrogels fromwater-soluble monomers by incorporating crosslinking monomers into thepolymer network. Typical examples are the range of hydrogels prepared bythe free-radical co-polymerization of acrylamide andN,N′-methylenebisacrylamide. Such hydrogels are relative to DIPS andTIPS more hydrophilic and more stable since the hydrophilic groups arean integral part of the polymer structure. It is well accepted that therange of monomers suitable for the production of such hydrogels israther limited, and is restricted to the requirement that both themonomer and the corresponding polymer need to be soluble in thepolymerization solvent.

To address this limitation, several attempts have been made to preparehydrogels by the bulk polymerization of monomers that produce waterinsoluble polymers. It is well accepted that the porosity of such gelsis dependent upon total monomer concentration of the reaction mixture.For example, hydrogels with higher total monomer content will have atighter network structure because of increased inter-penetration ofpolymer chains during network formation (Baker, J.; Hong, L.; Blanch,H.; Prausnitz, J. Macromolecules 1994, 27, 1446). As a result of this,and their high polymer content, hydrogels prepared in bulk are normallypoor in mechanical strength (glassy and brittle), low inbiocompatibility and water content, and possess a very limited pore sizerange. The absence of water in the synthesis environment of suchhydrogels also makes subsequent solvent exchange with water difficult.

Polymerization-induced phase separation (PIPS) is a process in which aninitially homogeneous solution of monomer and solvent becomes phaseseparated during the course of its polymerization. In hydrogelsynthesis, PIPS can be induced by a number of factors: continuousincrease in the fraction of molecules with high molecular weight, theunfavourable interactions between the polymer and other species in thereaction mixture, or the elasticity of the resultant polymeric network(Du{hacek over (s)}ek, K J. J. Polym. Sci. Polym. Symp. 1967, 16, 1289;Boots, H. M. J.; Kloosterboer, J. G.; Serbutoviez, C.; Touwslager, F. J.Macromolecules 1996, 29, 7683). Depending on the relative rates of thephase separation and the polymerization processes, PIPS can occur by themechanism of nucleation-growth in the metastable region, or by spinodaldecomposition in the multiphase coexisting region of the phase diagram(Eliçabe, G. E.; Larrondo, H. A.; Williams, R. J. J. Macromolecules1997, 30, 6550; Eliçabe, G. E.; Larrondo, H. A.; Williams, R. J. J.Macromolecules 1998, 31, 8173).

In the homo-polymerizations of a mono-vinyl monomer, during the courseof the reaction, because of the continuous increase in the fraction ofpolymer in the reaction mixture, PIPS can occur if the polymers formedin the reaction mixture are not miscible with the polymerizationsolvent. For example, PIPS occurs at ˜30% monomer conversion during thepolymerization of a mixture composed of 30% 2-hydroxyethyl methacrylateand 70% water when the molecular weight of the resultant polymer is˜300,000; and at ˜25% monomer conversion during the polymerization of amixture composed of 20% acrylamide, 32.5% poly(ethylene glycol)-400 whenthe molecular weight of the resultant polymer is ˜10,000.

Miscibility in a multi-component system is governed by its Gibbs freeenergy of mixing (ΔG_(mix)), which is a function of the enthalpies ofmixing and the entropies of mixing between the various components in themixture (ΔG_(mix)=ΔH_(mix)−TΔS_(mix)). Because the enthalpy of mixingbetween two chemically different polymers is mostly positive, increasesin the average molecular weight of the polymer solution will decreasethe overall entropy of the system. It is also expected to decrease themiscibility of the polymerization mixture. This leads to the occurrenceof PIPS at lower monomer conversions. For example, the onset of PIPS isat ˜1% monomer conversion during the polymerization of a mixturecomposed of 20% acrylamide, 32.5% poly(ethylene glycol)-400 when themolecular weight of the resultant polymer is ˜5,500,000. Polymer systemswith higher average molecular weight will be less miscible thancorrespond systems with lower average molecular weight.

In a simplified gel formation process by the free radicalco-polymerization of mono-vinyl monomer and multi-vinyl crosslinker,linear polymers are first formed in the solution during the fastpropagation step, and later crosslinked with other molecules in closeproximity by reaction through their pendent double bonds and additionalmonomer units (Stepto, R. F. T. “Non-linear polymerization, gelation andnetwork formation, structure and properties”, in Stepto, R. F. T. (ed.)Polymer Networks 1998; London, Blackie Academic & Professional, 14-63).Therefore, in a gel formation process, the average molecular weight ofthe polymer solution increases with increasing monomer conversionbecause of the ongoing crosslinking reactions.

Because hydrogels are defined as a network with infinite molecularweight which reaches the macroscopic dimensions of the sample itself(Flory P. J. Principles of polymer science. New York: Cornell UniversityPress, 1953 (Chapter IX)), polymers with very high molecular weight areproduced in the reaction mixture prior to the formation of a gelnetwork. Such polymers are therefore expected to undergo phaseseparations when the polymerization solvent is immiscible with theircorresponding linear polymer analogues with high molecular weight.

Acrylamide hydrogels, for separation in zone electrophoresis, wereintroduced in 1959 (Raymond, Weintraub, Science 1959, 130, 711) andwidely used as matrices for gels, and other electrophoretic operations.For example, one membrane-based electrophoresis technique (Gradiflow™(Gradipore, Australia)) involves a fixed boundary preparativeelectrophoresis method (U.S. Pat. No. 5,650,055, U.S. Pat. No. 5,039,386and WO 0013776) and utilizes a thin acrylamide hydrogel membrane with adefined pore size (D. B. Rylatt, M. Napoli, D. Ogle, A Gilbert, S. Lim,and C. H. Nair, J. Chromatog., A, 1999, 865, 145-153). However, despiteits widespread popularity, there are several potential hazards andlimitations which accompany the use of acrylamide hydrogel. For example,although the polymer is not toxic, exposure to the monomer andcrosslinker at manufacture during preparation of the gel posessignificant health concerns. In addition, residual and derivativechemical present in the gel may also pose potential health concern.

Currently, the pore size range of commercially available membranes issomewhat limited. For example, large pores suitable for DNA and RNAseparations are not routinely available. It is well known that for anacrylamide hydrogel, although an increase in pore size can be achievedby decreasing the polymer content, the mechanical strength and integritywill also be decreased. The loss of gel rigidity places a practicallimit on the accessible size separation range of a given material. Inorder to attempt to overcome these problems and to obtain matrices ofhigher porosity, Righetti (U.S. Pat. No. 5,785,832) and Uriel (U.S. Pat.No. 3,578,604) proposed polyacrylamide-agarose mixed-bed matrices. Thematrix was obtained by a simultaneous but independent process of agaroseand acrylamide gelification leading to an intertwining of the twopolymers. The agarose used, however, is normally based on naturallyoccurring raw materials which often have associated chemical andstructural impurities.

Righetti (U.S. Pat. No. 5,470,916) described a process for synthesisingpolyacrylamide matrixes with large pores. The process consists ofadding, to the polymerization monomer mixture, hydrophilic polymers(e.g. polyethylene glycol, polyvinylpyrrolidone, hydroxymethylcellulose) which, when added at a given concentration to the monomermixture, force the chains to agglomerate together, thus forming a gelnetwork having fibres of a much larger diameter than a regularacrylamide hydrogel. It was understood that the large pores were formeddue to the competition between gelation and phase separation in thesystem (Asnaghi, D., Giglio, M., Bossi, A., Righetti, P. G., J. Mol.Strut. 1996, 38, 37). It is, however, hard to control the ranges of poresize obtainable using this technique.

Another approach to the synthesis of hydrogels with large pores isprovided by template strategies (Beginn, U., Adv. Mater. 1998, 19, 16).This process resembles macroscopic metal casting processes in whichtemplates preform the shapes of the pores like casting-cores areintroduced into a liquid system and subsequently embedded by hardeningof the solvent (i.e. polymerization). After removal of these cores fromthe surrounding matrix the shape of the voids that remain reflects theform of the templates.

Rill et al. (Rill, R. L., Locke, B. R., Liu, Y., Dharia, J., Van Winkle,D. L., Electrophoresis 1996, 17, 1304; Rill, R. L., Van Winkle, D. L.,Locke, B. R., Anal. Chem. 1998, 70, 2433, Chakrapani, M., Van Winkle, D.H., Rill, R. L., Langmuir 2002, 18, 6449) reported templated acrylamidehydrogels as gel electrophoresis matrix and potential support for gelpermeation chromatography. They showed that templating gels with sodiumdodecyl sulfate (SDS) at concentrations up to 20% altered theelectrophoretic separations of SDS-protein complexes in a mannerconsistent with the creation of pores by SDS micelles. Anderson (U.S.Pat. No. 5,244,799) described a process in which templated hydrogelswere created by polymerizing a mixture of a hydrophilic monomer,polymerizing agent, an ionic surfactant and water. However, the usage ofsurfactants as template also have a few limitations, such as i) foamingproblems during the degassing and the polymerization process; ii) theneed to equilibrate the monomer solution (Method from Anderson involvethe equilibration of the monomer solution for at least a week); iii) insuch procedures, it is difficult to completely remove the ionicsurfactant from the hydrogel after the polymerization step. Andersondescribed an additional step in which the hydrogel was to be treatedwith a non-ionic surfactant solution while Rill et al. reported theremoval of 98% of SDS from the gel upon successive soaking in water.Residue ionic groups on the hydrogel matrix often caused undesirableelectroendosmotic properties when exposed to an electric field, and moreimportantly, were able to affect biomolecule separation by physicalinteractions with charged groups on them; and iv) high surfactantconcentrations are required to form the necessary interconnectingtemplating pores. At such concentrations, polyacrylamide is oftenincompatible with the ionic surfactant, resulting in undesirable phaseseparation during the polymerization. For example, Antonietti et al.(Antonietti, M., Caruso, R. A., Goltner, C. G., Weissenberger, M. C.Macromolecules 1999, 32 1383) reported during the formation of a varietyof polymer gels such as polyacrylamide in the presence of lyotropicsurfactant mesophases that “prior to polymerization all mixtures aretransparent, and become opaque or turbid-white shortly after the startof the reaction”. Rill also reported that gels formed in the presence of30% or more SDS became uniformly white as the surfactants were removed.

Undesirable swelling or shrinking has always been a drawback in the useof acrylamide hydrogels in non-aqueous operating systems such as theseparation of ions in non-aqueous systems and the electrophoreticseparation of hydrophobic proteins using organic solvents. Hydrogelssynthesised in a solvent similar to that of its final operatingenvironment will be more tolerant to solvent compositional changes.Typical solvents used in non-aqueous operating systems include alcohols,glycols, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO),tetramethylurea, formamide, tetramethylene sulfone, chloral hydrateN-methyl acetamide, N-methyl pyrollidone and phenol. It is, however,well known that when amounts of water-miscible solvents such as DMF,DMSO, TMU, ethylene glycol, or propylene glycol are added to theacrylamide polymerization mixture, the mechanical strength and clearnessof the polymer gel are severely compromised.

Amphiphilic polymer networks of α,ω-(meth)acryloyloxy monomers such aspoly(2-hydroxyethyl methacrylate) (poly(HEMA) have been studiedextensively as materials for pharmaceutical and biomedical applications,including carriers for controlled drug delivery and materials forprosthetic devices. The mechanical strength provided by the hydrophobicbackbone and the hydrophilicity of the hydroxy and ester groups on thepolymer side chains make polymers produced from HEMA excellentcandidates for hydrogels for separation processes. Zewert and Harrington(U.S. Pat. No. 5,290,411; U.S. Pat. No. 5,290,411; Zewert, T.,Harrington, M., Electrophoresis 1992, 13, 817-824; Zewert, T.,Harrington, M., Electrophoresis 1992, 13, 824), and Solomon et al. (PCTAU01/01632) have described the usage of hydrogels prepared fromα,ω-(meth)acryloyloxy monomers in various electrophoretic operations.

Most existing 2-Hydroxyethyl methacrylate (HEMA) systems are prepared inbulk, or with <50% diluent. Owing to the hydrophobicity of the network,organic diluents such as ethylene glycol and di(ethylene glycol) arenormally used (WO 00/44356; Caliceti, P., Veronese, F., Schiavon, O., IlFarmaco 1992, 47, 275, Carenza, M., Radiat. Phys. Chem. 1993, 42, 897).Although the properties of these hydrogels can be modified bycrosslinking or by the use of different diluents, their swelling inwater is thermodynamically limited to ˜40% (Havsky, M., Prins, W.,Macromolecules 1970, 3, 415; Nakamura, K., Nakagawa, T., Journal ofPolymer Science 1975, 13, 2299).

As a result, such HEMA hydrogels are normally poor in mechanicalstrength (glassy and brittle), low in biocompatibility, low in watercontent, and possess a very limited pore size range. The absence ofwater in the synthesis environment of such hydrogels also madesubsequent solvent exchange with water difficult. In addition, thetoxicity of some of the diluents is of great concern. Such hydrogelshave been predominantly used in applications that desire low waterswelling, such as contact lenses and transport membranes for gases andions (Corkhill, P. H., Jolly, A. M., Ng, C. O., Tighe, B. J. Polymer1987, 28, 1758; Hamilton, C. J., Murphy, S. M., Atherton, N. D., Tighe,B. J., Polymer 1988, 29, 1879).

It is well accepted that the porosity of such hydrogels is dependentupon the particular monomer, particular crosslinking agent, and thedegree of crosslinking. For example, hydrogels with higher total monomercontent will have a tighter network structure because of increasedinterpenetration of polymer chains during network formation (Baker, J.;Hong, L.; Blanch, H.; Prausnitz, J. Macromolecules 1994, 27, 1446). Itis thus highly desirable to be able to produce an HEMA hydrogel withhigh water content at a low initial concentration of monomers (<50 wt %)in order to obtain the desired biocompatibility and pore sizes forapplications such as electrophoresis separation membranes.

Several attempts have been made to improve the water swelling propertiesof HEMA hydrogels and to prepare such gel at a low initial concentrationof monomers.

i) HEMA hydrogels were synthesised in various hydro-organic solvents.Refojo (Refojo, M., Journal of Polymer Science: Part A-1 (1967), 5,3103) reported that visually clear hydrogels of poly(2-hydroxyethylmethacrylate) may be prepared by conducting the polymerization inethylene glycol-water solution. The phase separation limit for this typeof system was reported to be 45% of water in the reaction solution,allowing the total monomer concentrations to be decreased by thereplacement of monomers with diluent (Warren, T., Prins, W.,Macromolecules (1972), 5, 506). In addition to the fact that HEMAhydrogels prepared in such diluent were reported to exhibit a narrowrange of swelling at equilibrium in water (41% water) regardless of theinitial dilution of the monomer solution and relatively low level ofcrosslinking. Results from our laboratory have shown that thisseparation limit is highly dependent upon both the amount of crosslinkerand the choices of diluent in the reaction solution, with someformulations forming heterogeneous opaque polymer mass even when thewater content is below 45%. Zewert and Harrington (Zewert, T.,Harrington, M., Electrophoresis 1992, 13, 817) reported HEMA hydrogelsynthesis in aqueous sulfolane solution and concluded that HEMApolymerization is thoroughly incompatible with sulfolane even ifsulfolane concentrations are as low as 10%.

ii) Various HEMA derivatives such as the poly(alkylene glycol) esters ofacrylic or methacrylic acid (e.g. poly(ethylene glycol) methacrylate)were used instead of HEMA to prepare hydrogels with improved waterswelling properties. The disadvantages of such monomers is that they areexpensive and difficult to prepare. In addition, the pore size ofhydrogels prepared by these monomers is also limited because of theirlarge molecular weight, restricting the number of monomer unitsavailable in the monomer mixture.

iii) In order to obtain HEMA hydrogels with improved water swellingproperties, it is common to copolymerize HEMA with a hydrophilic monomersuch as acrylamide. Bajpai and Shrivastava (Bajpai, A. K., Shrivastava,M. J. Biomater. Sci. Polymer Edn 2002, 13, 237) copolymerised HEMA withacrylamide (% acrylamide>40 mol %) in the presence of a hydrophilicpolymer, poly(ethylene glycol) (PEG, MW 600). It was found that theswelling ratio of such hydrogel increases with increasing PEG 600content in the monomer mixture to a maximum at 4.31% (by weight). Suchhydrogels, according to the authors, “could be regarded as a network ofpoly(ethylene glycol) and poly(HEMA-co-acrylamide) chains thus creatingfree volumes of varying meshes for accommodating penetration of watermolecules”. It was also stated by Baipai and Shrivastava that there isno clear advantage of using a highly hydrophilic polymer content—“beyond0.56 of PEG (600) content (4.31%), the network density of the gel maybecame so high that mesh sizes of free volumes available between thenetwork chains get reduced . . . thus decreasing the swelling of thegel”. It is clear that the co-polymerization of acrylamide with HEMAdoes not eliminate the disadvantages associated with acrylamidehydrogels.

The present inventors have now developed new hydrogels suitable for anumber of separation techniques. The present invention also providesvisually clear hydrogels with good water compatibility and swellingproperties to be synthesized from monomers in hydro-organic or organicsolvents.

DISCLOSURE OF INVENTION

In a first aspect, the present invention provides a process forproducing a polymeric hydrogel having a network containing macroporesand micropores, the process comprising:

(a) forming a mixture by adding at least one monomer having at least onedouble bond, at least one crosslinker having at least two double bonds,an initiation system, and an organic additive to form a hydro-organicsystem with water; and

(b) allowing the monomer and crosslinker to copolymerize to form ahydrogel having a polymeric network containing macropores andmicropores.

The monomer having at least one double bond may be selected from polyolesters of acrylic or methacrylic acid, where the polyol is selected froma group which includes polyethylene glycol, a range of polyethyleneglycol esters or ethers, polypropylene glycol, a range of polypropyleneglycol esters or ethers, random or block copolymers of ethylene glycoland propylene glycol, or any suitable polyols such as glycerol,pentaerythritol, ethylene glycol or propylene glycol which are fully orpartly esterified. Mixtures consist of at least two of the abovemonomers can also be used.

Mixtures of the above monomer with any other well-known monomerssuitable for free radical polymerization may be used.

Preferably the monomer is used from about 1 to 80%, more preferably,from about 5 to 50%.

Preferably, the monomer is one or more hydrophilic monomers from theesters of acrylic or methacrylic acids.

In one preferred form, the monomer is hydroxyethyl methacrylate (HEMA).

The crosslinker having at least two double bond may be selected fromesters of acrylic and/or methacrylic acid, or acrylic or methacrylicacid with various polyols. Typical polyols include polyethylene glycol,a range of polyethylene glycol, a range of polypropylene glycol, randomor block copolymers of ethylene glycol and propylene glycol, or anysuitable polyols such as glycerol, pentaerythritol, ethylene glycol orpropylene glycol which may be partly esterified (for example, glycerolcan be esterified with two molecules of methacrylic acid to give thecrosslinking mixture). Mixtures consist of at least two of the abovecrosslinkers can also be used.

Mixtures of above crosslinker with any other well-known crosslinkerssuitable for free radical polymerization may be used.

Preferably use of the above crosslinker with greater than about 50% inthe mixture of crosslinkers; more preferably greater than about 80%.

In one preferred form, the crosslinker is ethylene glycol dimethacrylate(EGDMA).

Preferably, the polymeric hydrogel is made from a mixture of monomercontent of about 10 to 40%M and crosslinker of about 1 to 30%X beforepolymerization. When HEMA and EGDMA are used, the preferred compositionsof monomer mixture of HEMA with EGDMA are less than about 40% M and lessthan about 20%X, respectively. It will be appreciated, however, thatother concentrations can be used depending on the monomer andcrosslinker used.

Any suitable free radical producing method can be used as the initiationsystem. The initiation system is preferably formed by the redox, thermalor photo initiator/s. More preferably, the redox initiator is formed byammonium persulphate (APS) with N,N,N′,N′-tetramethylethylenediamine(TEMED).

The organic additive, which may be monomeric or polymeric (such asethylene glycol or polyethylene glycol), is preferably a hydrophilicpolymer miscible with water and miscible with a linear polymer producedfrom the monomer used for copolymerization; or a hydrophilic polymermiscible with water and has a similar solubility parameter(±10(MPa)^(0.5)) to that of a polymer produced from the monomer used forcopolymerization. The organic additive can be a single entity acting asboth a porogen to form macropores during the polymerization and asolvent with water to form the hydro-organic solvent.

The organic additive is preferably selected from ethylene glycol orpolyethylene glycol, propylene glycol or polypropylene glycol, random orblock copolymers of any of the above mixtures, or any of the aboveadditives that have an ester or ether end group. Mixtures consist of atleast two of the additives can also be used.

More preferably, the organic additive has the following generalformulation:

R₁, R₄=H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), —C(═O)—R₅ (R₅=(CH₂)_(x)—CH₃(x=0-4))

R₂, R₃=H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), OH

In a preferred from, the organic additive is a polyethylene glycol orpolypropylene glycol. The polyethylene glycol preferably has a molecularweight range from about 100 to 100000; preferably from about 200 to10000; more preferably from about 400 to 4000.

The polypropylene glycol typically has a molecular weight range fromabout 100 to 100000; preferably from 200 to 10000; more preferably fromabout 58 to 600.

In another preferred form, the organic additive is a copolymer with ahydrophilic component and a hydrophobic component. Preferably, theorganic additive is a copolymer of polyethylene glycol withpolypropylene glycol.

In use, the polymeric hydrogel formed can be used in the hydro-organicsolvent or the hydro-organic solvent components exchanged with water.

In a second aspect, the present invention provides a polymeric hydrogelhaving a network containing macropores and micropores produced by theprocess according to the first aspect of the present invention.

In a third aspect, the present invention provides a polymeric hydrogelcomprising a network of macropores and micropores formed bycopolymerizing at least one monomer having at least one double bond andat least one crosslinker having at least two double bonds in thepresence of a organic additive forming a hydro-organic system withwater.

The monomer having at least one double bond may be selected from polyolesters of acrylic or methacrylic acid, where the polyol is selected froma group which includes polyethylene glycol, a range of polyethyleneglycol esters or ethers, polypropylene glycol, a range of polypropyleneglycol esters or ethers, random or block copolymers of ethylene glycoland propylene glycol, or any suitable polyols such as glycerol,pentaerythritol, ethylene glycol or propylene glycol which are fully orpartly esterified. Mixtures consist of at least two of the abovemonomers can also be used.

Mixtures of the above monomer with any other well-known monomerssuitable for free radical polymerization may be used.

Preferably use of above monomer with greater than 50% in the mixture ofmonomers; more preferably greater than 80%.

Preferably, the monomer is one or more hydrophilic monomers from theesters of acrylic or methacrylic acids.

In one preferred form, the monomer is hydroxyethyl methacrylate (HEMA).

The crosslinker having at least two double bond may be selected fromesters of acrylic and/or methacrylic acid, or acrylic or methacrylicacid with various polyol. Typical polyols are polyethylene glycol, arange of polyethylene glycol, a range of polypropylene glycol, random orblock copolymers of ethylene glycol and propylene glycol, or anysuitable polyols such as glycerol, pentaerythritol, ethylene glycol orpropylene glycol which may be partly esterified (for example, glycerolcan be esterified with two molecules of methacrylic acid to give thecrosslinking mixture). Mixtures consist of at least two of the abovecrosslinkers can also be used.

Mixtures of above crosslinker with any other well-known crosslinkerssuitable for free radical polymerization may be used.

Preferably use of the above crosslinker with greater than 50% in themixture of crosslinkers; more preferably greater than 80%.

In one preferred form, the crosslinker is ethylene glycol dimethacrylate(EGDMA).

Preferably, the polymeric hydrogel is made from a mixture of monomercontent of about 10 to 40%M and crosslinker of about 1to 30%X beforepolymerization. When HEMA and EGDMA are used, the preferred compositionsof monomer mixture of HEMA with EGDMA are less than about 40% M and lessthan about 20% X. It will be appreciated, however, that otherconcentrations can be used depending on the monomer and crosslinkerused.

Any suitable free radical producing method can be used as the initiationsystem. The initiation system is preferably formed by the redox, thermalor photo initiator/s. More preferably, the redox initiator is formed byammonium persulphate (APS) with N,N,N′,N′-tetramethylethylenediamine(TEMED).

The organic additive, which may be monomeric or polymeric, is preferablya hydrophilic polymer miscible with water and miscible with a linearpolymer produced from the monomer used for copolymerization; or ahydrophilic polymer miscible with water and has a similar solubilityparameter (±10(MPa)^(0.5)) to that of a polymer produced from themonomer used for copolymerization. The organic additive can be a singleentity acting as both a porogen to form macropores during thepolymerization and a solvent with water to form the hydro-organicsolvent.

The organic additive is preferably selected from ethylene glycol orpolyethylene glycol, propylene glycol or polypropylene glycol, random orblock copolymers of any of the above mixtures, or any of the aboveadditives that have an ester or ether end group. Mixtures consist of atleast two of the additives can also be used.

More preferably, the organic additive has the following generalformulation:

R₁, R₄═H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), —C(═O)—R₅(R₅═(CH₂)_(x)—CH₃(x=0-4))

R₂, R₃H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), OH

In a preferred from, the organic additive is a polyethylene glycol orpolypropylene glycol. The polyethylene glycol preferably has a molecularweight range from about 100 to 100000; preferably from about 200 to10000; more preferably from about 400 to 4000.

The polypropylene glycol typically has a molecular weight range fromabout 100 to 100000; preferably from 200 to 10000; more preferably fromabout 58 to 600.

In another preferred form, the organic additive is a copolymer with ahydrophilic component and a hydrophobic component. Preferably, theorganic additive is a copolymer of polyethylene glycol withpolypropylene glycol.

Preferably, the mixture is degassed to remove any dissolved oxygen priorto polymerization.

In use, the polymeric hydrogel formed can be used in the hydro-organicsolvent or the hydro-organic solvent components exchanged with water.

In a fourth aspect, the present invention provides a separation mediumformed from the polymeric hydrogel according to the second or thirdaspects of the present invention.

Preferably, the separation medium is in the form of membrane, slab,beads or column. The medium is particularly suitable as anelectrophoretic medium capable of separating large biomolecules orcompounds having a molecular weight of at least 2000 k.

In a fifth aspect, the present invention provides a visually clearpolymeric hydrogel according to the second or thirds aspects of thepresent invention.

The present inventors have found that by the use of mixtures of waterand water-miscible entities as the polymerization solvent, visuallyclear hydrogels can be prepared even when the polymerization solvent isimmiscible with the corresponding linear polymer analogues. For example,a mixture of 20% poly(acrylamide)-5,500,000, 1% poly(vinylalchol)-18,000 (88% hydrolyzed), and 79% water is immiscible, but thepolymerization of 20% solutions of acrylamide andN,N′-methylenebisacrylamide can give visually clear gels; a mixture of15% poly(2-hydroxyethyl methacrylate)-300,000, 75% ethylene glycoldimethyl ether or 75% poly(ethylene glycol) dimethyl ether, and 10%water is immiscible, but the polymerization of 15% solutions of2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate in thesesolvents can give visually clear gels.

These results are new and unexpected because the general teaching frommost scientific literature on monomer selection for hydrogel synthesisis that the polymerization solvent should be a solvent for the linearanalogues of the resultant polymeric network.

By the selection of the water-miscible entities, the ‘freezing point’ ofthe reaction mixture can be controlled such that it occurs at a monomerconversion lower than the critical monomer conversion for the onset ofPIPS. The ‘freezing point’ of the reaction mixture is defined as thecritical monomer conversion at which the viscosity of the mixturereaches a specific level when the mobility of polymer chains in themixture becomes negligible and the dynamic concentration fluctuations ofpre-gel polymer solutions are frozen in the final network structure. Theresultant hydrogels of these systems will be visually clear and have arelatively uniform network because the polymer mixture was frozen in itsmiscible state before phase separation could occur. Hydrogels preparedby this approach have superior swelling, opitcal, and mechanicalproperties to that prepared by systems that reaches the phase boundarybefore the gel point. Those gels are formed from dispersions ofprecipitated polymers in the liquid phase (Okay O. Polymer 1999, 40,4117) and are highly opaque polymer masses that have very differentproperties from hydrogels synthesized using our approach.

In a sixth aspect, the present invention provides a method forseparating one or more compounds according to size usingelectrophoresis, the method comprising:

(a) providing a medium in the form of polymeric hydrogel having anetwork containing macropores and micropores according to the second orthird aspects of the present invention;

(b) adding one or more compounds to part of the medium; and

(c) applying an electric potential causing at least one compound to passthrough the medium, wherein movement through the medium is related tothe size of the compound.

In a seventh aspect, the present invention provides a size exclusionelectrophoresis system comprising:

(a) a cathode;

(b) an anode; and

(c) a separation medium in the form of polymeric hydrogel having anetwork containing macropores and micropores according to the second orthird aspects of the present invention capable of separating a mixtureof compounds according to size, the medium disposed between the anodeand cathode.

In a preferred form, the system further includes means for supplying asample containing one or more compounds to be separated to the system.

In a preferred form, the system further includes means for retaining orcapturing a compound separated by the system.

In a preferred form, the system further includes a voltage supply andmeans for applying an electric potential between the cathode and anode.

The system can be formed by having the separation medium disposedbetween two ion-permeable barriers forming two chambers either side ofthe size exclusion medium. Sample containing the compound(s) to beseparated can be placed in one of the chambers and, under the influenceof the applied voltage, a compound will move through the separationmedium in accordance with its size (large molecules elute out first) tothe second chamber where it can be retained or collected. It is alsopossible to have a plurality of different separation media disposedbetween the cathode and anode. In this form, preferably each separationmedium would have a different pore structure so as to be able toseparate compounds of different size.

In a eighth aspect, the present invention provides use of a separationmedium in the form of polymeric hydrogel having a network containingmacropores and micropores according to the second or third aspects ofthe present invention in size exclusion electrophoresis.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this application.

In order that the present invention may be more clearly understood,preferred forms will be described with reference to the followingdrawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows migration ratios of Kaleidoscope Pre-stained Standards in10%M 2%X acrylamide gel cassette synthesized in water, aqueous solutionsof ethylene glycol (25%) or propylene glycol (25%).

FIG. 2 shows migration ratios of SDS-PAGE Molecular Weight Standards(board range) in 10%M 2%X acrylamide gel cassette synthesized in water,or aqueous solutions of poly(ethylene glycol).

FIG. 3 shows migration ratios of SDS-PAGE Molecular Weight Standards(board range) in 10%M 2%X acrylamide gel cassette synthesized in wateror aqueous solutions of tri(ethylene glycol) and poly(ethylene glycol).

FIG. 4 shows migration ratios of Kaleidoscope Prestained Standards in10%M 2%X acrylamide gel cassette synthesized in water and aqueoussolutions of tri(ethylene glycol).

FIG. 5 shows turbidity results of polymers synthesized according toExample 29.

FIG. 6 shows turbidity results of polymers synthesized according toExample 30.

FIG. 7 shows turbidity results of polymers synthesized according toExample 31.

FIG. 8 shows turbidity results of polymers synthesized according toExample 32.

FIG. 9 shows turbidity results of polymers synthesized according toExample 33.

FIG. 10 shows turbidity results of polymers synthesized according toExample 34.

FIG. 11 shows turbidity results of polymers synthesized according toExample 35.

FIG. 12 shows turbidity results of polymers synthesized according toExample 36.

FIG. 13 shows turbidity results of polymers synthesized according toExample 37.

FIG. 14 shows turbidity results of polymers synthesized according toExample 38.

FIG. 15 shows the separation and migration pattern of Bovine serumalbumin (MW 67,000) by a 15%M 4%X HEMA/EGDMA membrane synthesized in 80%aqueous PEG 200 solution (Example 41) using 40 mM MES bis-TRIS buffer.

FIG. 16 shows turbidity results of polymers synthesized according toExample 56.

FIG. 17 shows a schematic diagram of the formation process of 20%Macrylamide hydrogels in the presence of water and a water-solubleentity. Line E represents systems with 0%X; line F, 2%X; line G, 3%X;line H, 10%X.

FIG. 18 shows real-time viscosity measurements of the polymerization of20%M acrylamide solutions, in the presence of 17.5% PEG-400, at various%X. Time at which phase separation was observed in the samples arerepresented by dark coloured points (circle).

FIG. 19 shows turbidity measurements of 20%M 2%X acrylamide hydrogelssynthesized in the presence of various amounts of PEG-400.

FIG. 20 shows the critical propylene glycol concentrations for theformation of visually hydrogels at various %M and %X.

FIG. 21 shows real-time viscosity measurements of the polymerization of20%M 2%X HEMA solutions in the presence of various amounts of propyleneglycol. Times at which phase separation was observed in the samples arerepresented by dark coloured points (circle)

FIG. 22 shows SEM images (10,000×) of cross-sectional interior ofswollen 10%M 2%X acrylamide hydrogels synthesized in water (A), 50%ethylene glycol solution (B), and 50% propylene glycol solution (C).

MODE(S) FOR CARRYING OUT THE INVENTION

Novel formulations for HEMA hydrogel synthesis

The present inventors have developed a new synthesis method using amixture of water and water-miscible entities as the polymerizationsolvent such that HEMA hydrogels can be crosslinked with ethylene glycoldimethacryate (EGDMA) using low initial monomer content (5-50%). Usingwater-miscible entities such as polymers with repeating ethoxylated andpropyoxylated units (e.g. poly(ethylene glycol) and poly(propyleneglycol) or random or block copolymers of poly(ethylene glycol) at apolymeric-additive glycol-water ratio of about 9:1 to 1:9), hydrogelsbased on HEMA were successfully formed having higher water swellingproperties and bigger pore sizes than those produced previously. Suchhydrogels can be subsequently used as synthesized or after thewater-miscible entities have been displaced with water. This result isunexpected, given that it is well known that high concentrations ofhydrophilic polymer (i.e. poly(ethylene glycol) and poly(propyleneglycol)) in acrylamide hydrogel synthesis would lead to phase separationof the reaction mixture. For example, Righetti (Righetti, P. GChromatogr. A 1995, 698, 3) observed that when acrylamide hydrogels weresynthesised in the presence of PEG 2000-20,000, turbid gels (phaseseparation) were produced and was a function of both length andconcentration of the polymer. It was observed that longer polymer chainsinduce phase separation at lower concentration; all gels become turbidwhen the PEG concentration in the solvent exceed 10 wt %.

It was also discovered by the present inventors that as the molecularweight of the water-miscible entities increases, the pore size of thehydrogels becomes dependent upon the properties of the entities, withthe entities acting as a “template”. In high molecular weight solvents,hydrogels synthesized in solutions of high molecular weight entitieswere observed to swell more than that of lower molecular weight. To ourknowledge, this is the first system in which the templating system isalso acting as the solvent for the hydrogel.

Multimodal hydrogels

Utilising the templating and the solvent properties of thewater-miscible entities, it was discovered that multimodal HEMAhydrogels can be obtained by careful selection of the concentrations ofmonomer, the crosslinking extent, and the types and concentrations ofwater-miscible entities in a one-step process. Two general types ofpores exist in such membranes—macropores formed by the template, andmicropores formed by the crosslinking of polymer chains. Dependent uponthe concentrations of the water-miscible entities, the macropores in thehydrogel can be continuous (i.e. interconnected), or non-continuous.

Derivatives of monomers such as the poly(alkylene glycol) esters ofacrylic or methacrylic acid can also be used in the same manner as HEMAto prepare hydrogels with multimodal channels.

Such hydrogels are different from these synthesised by Zewert andHarrington (U.S. Pat. No. 5,290,411 and U.S. Pat. No. 5,290,411)because:

i) Their teaching indicates that the pore size of the gel is dependentupon the types and concentration of monomer and crosslinkers. Pore sizesof hydrogels according to the present invention are not only dependentupon the types and concentration of monomer and crosslinkers but alsodependent upon the size of the water-miscible entities;

ii) The present hydrogels have two types of pores within its network,macropores and micropores;

iii) In the patent of Zewert and Harrington, organic solvents were addedmainly for the usage of the resultant gel in organic electrophoresis andwere not subsequently replaced with water. In the present invention, thewater-miscible entities are acting both as a solvent and a template, andare subsequently exchanged with water.

Applications

HEMA hydrogels made with the above formulations are particularlywell-suited for use as separation membranes for biomolecules. Otherrelated areas of interest include biocompatible applications such asprosthetic devices, drug release matrixes, and tissue scaffolds.

Membrane-Based Electrophoresis

A number of membrane-based electrophoresis apparatus developed byGradipore Limited, Australia were used in the following experiments. Insummary, the apparatus typically included a cartridge which housed anumber of membranes forming two chambers, cathode and anode connected toa suitable power supply, reservoirs for samples, buffers andelectrolytes, pumps for passing samples, buffers and electrolytes, andcooling means to maintain samples, buffers and electrolytes at arequired temperature during electrophoresis.

The cartridge contained three substantially planar membranes positionedand spaced relative to each other to form two chambers through whichsample or solvent can be passed. A separation membrane was positionedbetween two outer membranes (termed restriction membranes as theirmolecular mass cut-offs are usually smaller than the cut off of theseparation membrane). When the cartridge was installed in the apparatus,the restriction membranes were located adjacent to an electrode. Thecartridge is described in AU 738361, which description is incorporatedherein by reference.

Description of membrane-based electrophoresis can be found in U.S. Pat.No. 5,039,386 and U.S. Pat. No. 5,650,055 in the name of GradiporeLimited, which description is incorporated herein by reference.

Polyacrylamide Gel Electrophoresis (PAGE)

Standard PAGE methods were employed as set out below.

Reagents: 10× SDS Glycine running buffer (Gradipore Limited, Australia),dilute using Milli-Q water to 1× for use; 1× SDS Glycine running buffer(29 g Trizma base, 144 g Glycine, 10 g SDS, make up in RO water to 1.0l); 10× TBE II running buffer (Gradipore), dilute using Milli-Q water to1× for use; 1× TBE II running buffer (10.8 g Trizma base, 5.5 g Boricacid, 0.75 g EDTA, make up in RO water to 1.0 l); 2× SDS sample buffer(4.0 ml, 10% (w/v) SDS electrophoresis grade, 2.0 ml Glycerol, 1.0 ml0.1% (w/v) Bromophenol blue, 2.5 ml 0.5M Tris-HCl, pH 6.8, make up in ROwater up to 10 ml); 2× Native sample buffer (10% (v/v) 10× TBE II, 20%(v/v) PEG 200, 0.1 g/l Xylene cyanole, 0.1 g/l Bromophenol blue, make upin RO water to 100%); Coomassie blue stain (Gradipure™, GradiporeLimited). Note: contains methanol 6% Acetic Acid solution for de-stain.

Molecular weight markers (Recommended to store at −20° C.): SDS PAGE(e.g. Sigma wide range); Western Blotting (e.g. color/rainbow markers).

SDS PAGE with non-reduced samples

To prepare the samples for running, 2× SDS sample buffer was added tosample at a 1:1 ratio (usually 50 μl/50 μl) in the microtiter platewells or 1.5 ml tubes. The samples were incubated for 5 minutes atapproximately 100° C. Gel cassettes were clipped onto the gel supportwith wells facing in, and placed in the tank. If only running one gel ona support, a blank cassette or plastic plate was clipped onto the otherside of the support Sufficient 1× SDS glycine running buffer was pouredinto the inner tank of the gel support to cover the sample wells. Theouter tank was filled to a level approximately midway up the gelcassette. Using a transfer pipette, the sample wells were rinsed withthe running buffer to remove air bubbles and to displace any storagebuffer and residual polyacrylamide.

Wells were loaded with a minimum of 5 μl of marker and the preparedsamples (maximum of 40 μl). After placing the lid on the tank andconnecting leads to the power supply the gel was run at 150V for 90minutes. The gels were removed from the tank as soon as possible afterthe completion of running, before staining or using for anotherprocedure (e.g. Western blot).

Staining and De-Staining of Gels

The gel cassette was opened to remove the gel which was placed into acontainer or sealable plastic bag. The gel was thoroughly rinsed withtap water, and drained from the container. Coomassie blue stain(approximately 100 ml Gradipure™, Gradipore Limited, Australia)) wasadded and the container or bag sealed. Major bands were visible in 10minutes but for maximum intensity, stained overnight. To de-stain thegel, the stain was drained off from the container.

The container and gel were rinsed with tap water to remove residualstain. 6% acetic acid (approximately 100 ml) was poured into thecontainer and sealed. The de-stain was left for as long as it takes toachieve the desired level of de-staining (usually 12 hours). Once at thedesired level, the acetic acid was drained and the gel rinsed with tapwater.

Size exclusion electrophoresis

Compared to column chromatography, which normally involve high pressuredrops and compaction for soft gels at high flow rates, membranechromatography has a lower pressure drop, high flow rate and highproductivity as result of microporous/macroporous structures inrelatively thin membranes.

As described above, protein separations under electrophoresis with aseparation membrane are normally either size or charge based, which havelimitations of its own such as the range of proteins can be separated.The present inventors have introduced the concept of protein or othercompound separation under size exclusion chromatography principle usingelectrophoresis. By using this concept, protein or compound can beseparated in an opposite manner to conventional electrophoresis and somelarge biomolecules, which are not able to be separated by existingsystems, have been purified by this process.

The basic requirements for a SE separation are that the separationmedium contains at least two types of pores: macropores and micropores.In chromatography, the large molecules will go though the big pores andtravel fast while the smaller molecules will have interaction with smallpores due to its compatible size with the micropores. Therefore in theseparation of polymers by using size exclusion chromatography, polymerwith largest molecular weight will elute out of a separating columnfirst and the one with the smallest molecular weight will elute outlast.

In the design of the SE hydrogel matrix systems, the present inventorshave adopted the same principle. The solvent system used can act both asa porogen and a solvent to the amphiphilic monomer. The monomers usedproduce network structures with functional groups and these functionalgroups can interact with small proteins as these molecules enter thesmall pore structure.

The hydrogels can be used in two different ways by utilizing therecently developed Gradiflow™ system to test the separation of theresultant membranes; one way is for the manufacture of membranes with alarger pore size or with improved functionality. The other is SEhydrogel electrophoresis.

Membranes with larger pore size can be tested in the following way: themembrane will be placed in the middle of a separation cartridge in aseparation unit. The protein mixture to be separated will be placed instream 1. When the charge is applied, the separation will begin andsmall proteins will travel to downstream through the membranes.

When SE type membrane is used, it is placed in the middle of aseparation cartridge in a separation unit. The protein mixture to beseparated will be placed in stream 1. When the electric potential isapplied, the separation will begin and large proteins will travel todownstream through the SE-type membranes. With the increase of time,small proteins may saturate the small pores of the separation membraneand the process needs to be pulsed to release the small proteins back tothe upstream. This process can be carried out by removing separatedproteins from downstream and reverse the potential supplied.

DEFINITIONS

The following terms shall have the indicated definitions unlessotherwise indicated:

“Hydrogel” is a chemically crosslinked polymer characterized byhydrophilicity and insolubility in water.

“Micropores” are pores within the gel network of the background matrix.The size of these pores can be related to the hydrogel formation speciesin the initial pre-gelling mixture using relationships and theoriesdeveloped for common electrophoretic matrixes. For example, microporeswithin an acrylamide hydrogel are related to the total monomerconcentration and monomer to crosslinker ratios in the free radicalpolymerization of acrylamide and N,N′-methylenebisacrylamide (Bansil,R.; Gupta, M. Ferroelectrics 1980, 30, 64).

“Macropores” are pores within the membrane that are significantly larger(more than 2 times) than micropores of the background matrix.

“Microporous membranen” is a separation membrane having substantiallycontinuous interconnecting micropores. Such membranes are usedextensively in preparative electrophoresis.

“Macroporous membrane” is a separation membrane having continuousinterconnecting micropores but non-continuous macropores (i.e.macropores are not connected directly to each other). Such membraneshave similar sieving properties to the corresponding microporousmembrane, but allows for higher flow rate through the matrix because ofthe reduced diffusional constraints.

“Size exclusion membrane (SE-Mem)” is a bi, or multimodal separationmembrane having continuous interconnecting micropores, andinterconnecting macropores within its matrix. SE-Mem can have differentseparation behaviours depending upon the size of the micropores(S_(mic)), the size of the macropores (S_(mac)) and the size of thebio-molecule mixture (S_(bio)). When S_(bio)>S_(mac)>S_(mic), noseparation would occur, when S_(mac)˜S_(bio)>S_(mic), all molecules withdimension smaller than the macropores would be separated from theirbigger counter part, when S_(mac)>S_(bio)˜S_(mic), all molecules withdimension smaller than the macropores would be separated from theirbigger counter part, and be eluted in the order of decreasing size.

From the above description of SE-Mem, the challenge in producing suchmembrane lies in i) increase the size exclusion limit, i.e. the size ofthe largest interconnecting pores, and ii) produce a polymer with bothinterconnecting micropores and macropores. It would be a substantialadvantage to develop a simple process to synthesis such membrane.

Multi-modal HEMA hydrogels are suitable to be used as SE-Mem as twogeneral types of pores exist in such membrane - macropores formed by thetemplate or porogen, and micropores formed by the crosslinking ofpolymer chains. The size exclusion limit of such membrane is alsoincreased because of the macropores.

SE-Mem can be used in membrane based electrophoresis techniques and asmembrane support for membrane chromatography and affinity membranechromatography. It can take the form of flat sheet, stacked sheet,radical flow cartridges, hollow fibre molecules, slab, and column.

The term “stream 1 (S1)” refers to denote the first interstitial volumewhere sample is supplied in a stream to the electrophoresis apparatus.This stream may also be called the “upstream”.

The term “stream 2 (S2)” is used in this specification to denote thesecond interstitial volume where material is moved from the firstinterstitial volume through the separation membrane to a stream of theelectrophoresis apparatus. This stream may also be called the“downstream”.

The term “forward polarity” is used when the first electrode is thecathode and the second electrode is the anode in the electrophoresisapparatus and current is applied accordingly.

The term “reverse polarity” is used when polarity of the electrodes isreversed such that the first electrode becomes the anode and the secondelectrode becomes the cathode.

ABBREVIATIONS

Acrylamide (AAm); N,N′-methylenebisacrylamide (BIS); poly(acrylamide)gel electrophoresis (PAGE); 2-hydroxyethyl acrylate (HEA);2-hydroxyethyl methacrylate (HEMA); poly(ethylene glycol) acrylate(PEGA); poly(ethylene glycol) methacrylate (PEGMA); ethylene glycoldiacrylate (EGDA); ethylene glycol dimethacrylate (EGDMA); poly(ethyleneglycol) acrylate (PEGA); poly(ethylene glycol) methacrylate (PEGMA);poly(ethylene glycol) diacrylate (PEGDA); poly(ethylene glycol)dimethacrylate (PEGDMA); poly(ethylene glycol) PEG; and poly(propyleneglycol) PPG; poly(ethylene glycol) methyl ether PEGME;N,N,N′N′-tetramethylethylenediamine (TEMED); ammonium persulfate (APS).

EXAMPLES Example 1 Preparation of Monomer Solutions

Two terms are introduced to classify the monomer solutions:

%M refers to the total concentration of monomer as a weight percentage;%X refers to the number of double bonds on the crosslinkers as a portionof the total number of double bonds on the monomers.${\%\quad M} = {\frac{{total}\quad{mass}\quad{of}\quad{monomers}\quad(g)}{{mass}\quad{of}\quad{reaction}\quad{mixture}\quad(g)} \times 100}$${\%\quad X} = {\frac{{number}\quad{of}\quad{double}\quad{bonds}\quad{on}\quad{crosslinkers}\quad({mol})}{{total}\quad{numbers}\quad{of}\quad{double}\quad{bonds}\quad{on}\quad{monomers}\quad({mol})} \times 100}$Preparation of Acrylamide Hydrogels

Example 2 Preparation of 10%M 2%X AAm/BIS hydrogels for swelling testsusing water as solvent

Monomer solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in water (9 g) in disposable glass vials. The monomersolution was then degassed by argon purging for 5 min prior to theaddition of the initiator system (0.2 mol % initiator per double bond)composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 3 Preparation of 10%M 2%X AAm/BIS hydrogels for swelling testsusing aqueous ethylene glycol as solvent

Aqueous solutions of ethylene glycol (25, 50 and 75%) were prepared byvarying amounts of ethylene glycol and water. AAm (978.3 mg) and BIS(21.7 mg) were added to the above solutions (9 g) in disposable glassvials. The monomer solution was then degassed by argon purging for 5 minprior to the addition of the initiator system (0.2 mol % initiator perdouble bond) composed of freshly made up 10% (w/v) APS and 10% (v/v)TEMED. The polymerization was then allowed to proceed at roomtemperature overnight under an argon environment.

Example 4 Preparation of 10%M 2%X AAm/BIS hydrogels for swelling testsusing aqueous propylene glycol as solvent

Aqueous solutions of propylene glycol (25, 50 and 75%) were prepared byvarying amounts of ethylene glycol and water. AAm (978.3 mg) and BIS(21.7 mg) were added to the above solutions (9 g) in disposable glassvials. The monomer solution was then degassed by argon purging for 5 minprior to the addition of the initiator system (0.2 mol % initiator perdouble bond) composed of freshly made up 10% (w/v) APS and 10% (v/v)TEMED. The polymerization was then allowed to proceed at roomtemperature overnight under an argon environment.

Example 5 Preparation of 10%M 2%X AAm/BIS hydrogels for swelling testsusing aqueous tri(ethylene glycol) as solvent

Aqueous solutions of triethylene glycol (22, 44, 67 and 72%) wereprepared by varying amounts of triethylene glycol and water. AAm (978.3mg) and BIS (21.7 mg) were added to the above solutions (9 g) indisposable glass vials. The monomer solution was then degassed by argonpurging for 5 min prior to the addition of the initiator system (0.2 mol% initiator per double bond) composed of freshly made up 10% (w/v) APSand 10% (v/v) TEMED. The polymerization was then allowed to proceed atroom temperature overnight under an argon environment.

Example 6 Preparation of 10%M 2%X AAm/BIS hydrogels for swelling testsusing aqueous poly(ethylene glycol) 400 as solvent

Aqueous solutions of poly(ethylene glycol) 400 (6, 11, 16 and 22%) wereprepared by varying amounts of poly(ethylene glycol) 400 and water. AAm(978.3 mg) and BIS (21.7 mg) were added to the above solutions (9 g) indisposable glass vials. The monomer solution was then degassed by argonpurging for 5 min prior to the addition of the initiator system (0.2 mol% initiator per double bond) composed of freshly made up 10% (w/v) APSand 10% (v/v) TEMED. The polymerization was then allowed to proceed atroom temperature overnight under an argon environment.

Example 7 Preparation of 10%M 2%X AAm/BIS hydrogels for turbiditymeasurements using aqueous tri(ethylene glycol) as solvent

Aqueous solutions of tri(ethylene glycol) (11, 22, 33, 44, 55, 61, 64,66, 69 and 72%) were prepared by varying amounts of tri(ethylene glycol)and water. AAm (978.3 mg) and BIS (21.7 mg) was added to the abovesolutions (9 g) in disposable glass vials. The monomer solution was thendegassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 8 Preparation of 10%M 2%X AAm/BIS hydrogels for turbiditymeasurements using aqueous poly(ethylene glycol) 400 as solvent

Aqueous solutions of poly(ethylene glycol) 400 (6, 11, 16, 19, 22, 27and 33%) were prepared by varying amounts of poly(ethylene glycol) 400and water. AAm (978.3 mg) and BIS (21.7 mg) were added to the abovesolutions (9 g) in disposable glass vials. The monomer solution was thendegassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 9 Preparation of 10%M 2%X AAm/BIS hydrogels for turbiditymeasurements using aqueous poly(ethylene glycol) 400 as solvent at 40°C.

Aqueous solutions of poly(ethylene glycol) 400 (6, 11, 16, 19, 22, 27and 33%) were prepared by varying amounts of poly(ethylene glycol) 400and water. AAm (978.3 mg) and BIS (21.7 mg) were added to the abovesolutions (9 g) in disposable glass vials. The monomer solution was thenplaced in a 40° C. water bath for 15 mins and degassed by argon purgingfor 5 min prior to the addition of the initiator system (0.2 mol %initiator per double bond) composed of freshly made up 10% (w/v) APS and10% (v/v) TEMED. Two 375 μl samples were pipetted into disposablecuvettes (10×10×45 mm³) and the polymerization was then allowed toproceed at 40° C. for 2 hr under an argon environment.

Example 10 Preparation of 10%M 2%X AAm/BIS hydrogels for turbiditymeasurements using aqueous poly(ethylene glycol) 20,000 as solvent

Aqueous solutions of poly(ethylene glycol) 20,000 (0.02, 0.04, 0.06,0.08, 0.1, 0.12 and 0.14%) were prepared by varying amounts ofpoly(ethylene glycol) 20,000 and water. AAm (978.3 mg) and BIS (21.7 mg)were added to the above solutions (9 g) in disposable glass vials. Themonomer solution was then degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two375 μl samples were pipetted into disposable cuvettes (10×10×45 mm³) andthe polymerization was then allowed to proceed at room temperatureovernight under an argon environment.

Example 11 Preparation of 10%M 2%X AAm/BIS hydrogel cassettes for gelelectrophoresis using water as solvent

10%M 2%X solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in water (6.5 g) and 1.5M Tris-HCl buffer (pH 8.8, 2.5 g).The stock buffer solution was prepared by dissolving Tris (27.23 g) inwater (80 ml) and adjusted to the pH of 8.8 with 6 N HCl followed bymaking up the required volume (150 ml) with water.

The monomer solution was degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS (64.1 μm) and 10% (v/v)TEMED (42.4 μm). The gel solution (7 ml) was then immediately castbetween two glass plates (8×8 cm, 1 mm apart) that were purged withargon and left to polymerize at room temperature for 3 hr under an argonenvironment prior to use.

Example 12 Preparation of 10%M 2%X AAm/BIS hydrogel cassettes for gelelectrophoresis using 25% aqueous ethylene glycol as solvent

10%M 2%X solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in ethylene glycol (2.7 g) and water (3.8 g). 1.5MTris-HCl buffer (pH 8.8, 2.5 g). The stock buffer solution was preparedby dissolving Tris (27.23 g) in water (80 ml) and adjusted to the pH of8.8 with 6 N HCl followed by making up the required volume (150 ml) withwater.

The monomer solution was degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS (64.1 μm) and 10% (v/v)TEMED (42.4 μm). The gel solution (7 ml) was then immediately castbetween two glass plates (8×8 cm, 1 mm apart) that were purged withargon and left to polymerize at room temperature for 3 hr under an argonenvironment prior to use.

Example 13 Preparation of 10%M 2%X AAm/BIS hydrogel cassettes for gelelectrophoresis using 25% aqueous propylene glycol as solvent

10%M 2%X solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in propylene glycol (2.7 g) and water (3.8 g). 1.5MTris-HCl buffer (pH 8.8, 2.5 g). The stock buffer solution was preparedby dissolving Tris (27.23 g) in water (80 ml) and adjusted to the pH of8.8 with 6 N HCl followed by making up the required volume (150 ml) withwater.

The monomer solution was degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS (64.1 μm) and 10% (v/v)TEMED (42.4 μm). The gel solution (7 ml) was then immediately castbetween two glass plates (8×8 cm, 1 mm apart) that were purged withargon and left to polymerize at room temperature for 3 hr under an argonenvironment prior to use.

Example 14 Preparation of 10M 2X AAm/BIS hydrogel cassettes for gelelectrophoresis using 11% aqueous tri(ethylene glycol) as solvent

10%M 2%X solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in tri(ethylene glycol) (1.2 g) and water (5.3 g). 1.5MTris-HCl buffer (pH 8.8, 2.5 g). The stock buffer solution was preparedby dissolving Tris (27.23 g) in water (80 ml) and adjusted to the pH of8.8 with 6 N HCl followed by making up the required volume (150 ml) withwater.

The monomer solution was degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator, per doublebond) composed of freshly made up 10% (w/v) APS (64.1 μm) and 10% (v/v)TEMED (42.4 μm). The gel solution (7 ml) was then immediately castebetween two glass plates (8×8 cm, 1 mm apart) that were purged withargon and left to polymerize at room temperature for 3 hr under an argonenvironment prior to use.

Example 15 Preparation of 10%M 2%X AAm/BIS hydrogel cassettes for gelelectrophoresis using 5.5 and 11% aqueous poly(ethylene glycol) 400 assolvent

10%M 2%X solutions (10 g) were prepared by dissolving AAm (978.3 mg) andBIS (21.7 mg) in poly(ethylene glycol) 400 (0.6 or 1.2 g) and water (5.3or 5.9 g). 1.5M Tris-HCl buffer (pH 8.8, 2.5 g). The stock buffersolution was prepared by dissolving Tris (27.23 g) in water (80 ml) andadjusted to the pH of 8.8 with 6 N HCl followed by making up therequired volume (150 ml) with water.

The monomer solution was degassed by argon purging for 5 min prior tothe addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS (64.1 μm) and 10% (v/v)TEMED (42.4 μm). The gel solution (7 ml) was then immediately castbetween two glass plates (8×8 cm, 1 mm apart) that were purged withargon and left to polymerize at room temperature for 3 hr under an argonenvironment prior to use.

Evaluation of Acrylamide Hydrogels

Swelling Tests

Gels made according to Examples 2-6 were immersed in water (500 g) for 1week during which the immersing solution (water) was exchanged on adaily basis. The gel was then dried in a 40° C. oven for 1 week. Theequilibrium solvent content of the gel was determined by the followingequation.${{Equilibrium}\quad{solvent}\quad{{content}({ESC})}} = \frac{{{weight}( {{swollen}\quad{gel}} )} - {{weight}( {{dried}\quad{gel}} )}}{{weight}( {{dried}\quad{gel}} )}$

Example 16 ESC (Water) of AAm/BIS hydrogels synthesized in water andaqueous solutions of ethylene glycol

Polymerization Solvent ESC water 12.1 25% ethylene glycol/75% water 14.350% ethytene glycol/50% water 15.4 75% ethylene glycol/25% water 20.0

Example 17 ESC (water) of AAm/BIS hydrogels synthesized in water andaqueous solutions of propylene glycol

Polymerization Solvent ESC water 12.1 25% propylene glycol/75% water15.3 50% propylene glycol/50% water 21.9 75% propylene glycol/25% water28.6

Example 17 ESC (water) of AAm/BIS hydrogels synthesized in water andaqueous solutions of tri(ethylene glycol)

Polymerization Solvent ESC water 12.1 11% tri(ethylene glycol)/89% water12.7 22% tri(ethylene glycol)/78% water 14.5 33% tri(ethyleneglycol)/66% water 16.3 44% tri(ethylene glycol)/56% water 18.1 55%tri(ethylene glycol)/45% water 21.8 61% tri(ethylene glycol)/39% water25.0 64% tri(ethylene glycol)/36% water 26.3 66% tri(ethyleneglycol)/34% water 26.7 69% tri(ethylene glycol)/31% water 30.0 72%tri(ethylene glycol)/28% water 32.8

Example 17 ESC (water) of AAm/BIS hydrogels synthesized in water andaqueous solutions of poly(ethylene glycol) 400

Polymerization Solvent ESC water 12.1  6% poly(ethylene glycol) 400/94%water 13.2 11% poly(ethylene glycol) 400/89% water 14.0 16%poly(ethylene glycol) 400/84% water 15.2 22% poly(ethylene glycol)400/78% water 17.3Turbidity Measurements

The turbidity of gels made according to examples 7-9 was measured usingUV-visible spectrophotometry. Distilled water was used for the baselineand the absorbance of each gel sample and the correspondingpolymerization solvent were recorded at 100 nm intervals between 300 and800 nm. The turbidity of the gel samples were determined by thefollowing equation.Turbidity=−log_(e)(10^(−(absorbance of gel−absorbance of polymerization solvent)))

Example 18 Turbidity of 10%M 2%X AAm/BIS hydrogels synthesized in waterand aqueous solutions of poly(ethylene glycol) 400 at 500 nm (roomtemperature and 40° C.)

Turbidity (Room Turbidity Polymerization Solvent Temperature) (40° C.)Water 0 0  6% poly(ethylene glycol) 400/94% water 0 0 11% poly(ethyleneglycol) 400/89% water 0 0 16% poly(ethylene glycol) 400/84% water 0.23 019% poly(ethylene glycol) 400/81% water 0.46 0.18 22% poly(ethyleneglycol) 400/78% water 1.27 0.32 27% poly(ethylene glycol) 400/73% water6.90 5.4 33% poly(ethylene glycol) 400/66% water 8.06 7.5*Visual opacity corresponds to a turbidity value of >0.3 at 500 nm

Example 19 Turbidity of 10%M 2% AAm/BIS hydrogels synthesized in waterand aqueous solutions of tri(ethylene glycol), poly(ethylene glycol) 400and poly(ethylene glycol 20,000 at 500 nm (room temperature)

Turbidity testing showed that the onset of opacity occurs at 72%, 19%and 0.1% for aqueous solution of tri(ethylene glycol), poly(ethyleneglycol) 400 and poly(ethylene glycol) 20,000 respectively.

Gel Electrophoresis

Standard SDS-PAGE was performed on the acrylamide hydrogel cassette(example 11-15) using a constant voltage of 150 V and Tris-glycineelectrophoresis running buffer. The electrophoresis running buffer (100ml) was prepared by dissolving Tris (9 g), SDS (3 g), and glycine (43.2g) in water and diluting 1:5 with water before use. 10 μL ofKaleidoscope pre-stained protein marker or SDS-PAGE molecular weightstandards (broad range) was syringed into sample wells and separated.Gels with SDS-PAGE molecular weight standards (broad range) were stainedfor 3 hr using Coomassie Blue solution and de-stained overnight with 10%aqueous acetic acid. The migration ratio of a protein was determined bythe following equation.${{Migration}\quad{Ratio}} = \frac{{distance}\quad{travelled}\quad{by}\quad{protein}}{{disctance}\quad{travelled}\quad{by}\quad{dyefront}}$Kaleidoscope Prestained Standards (Bio-Rad 161-0324) Protein CalibratedMW Myosin 206,000 β-galactosidase 128,000 Bovine serum albumin 81,000Carbonic anhydrase 40,300 Soybean trypsin inhibitor 31,600 Lysozyme19,300 Aprotinin 7,800

SDS-PAGE Molecular Weight Standards (board range, Bio-Rad 161-0317)Protein Calibrated MW Myosin 200,000 β-galactosidase 116,250Phosphorylase b 97,400 Serum albumin 66,200 Ovalbumin 45,000 Carbonicanhydrase 31,000 Trypsin inhibitor 21,500 Lysozyme 14,400 Aprotinin6,500

Example 20 Electrophoresis of 10%M 2%X AAm/BIS gel cassette synthesizedin water and aqueous solutions of ethylene glycol or propylene glycol

Migration ratios of Kaleidoscope Pre-stained Standards in 10%M 2%Xacrylamide gel cassette synthesized in water, aqueous solutions ofethylene glycol (25%) or propylene glycol (25%) are shown in FIG. 1.

Example 21 Electrophoresis of 10%M 2%X acrylamide gel cassettesynthesized in water and aqueous solutions of poly(ethylene glycol) 400

Migration ratios of SDS-PAGE Molecular Weight Standards (board range) in10%M 2%X acrylamide gel cassette synthesized in water, or aqueoussolutions of poly(ethylene glycol) are shown in FIG. 2.

Example 22 Electrophoresis of 10%M 2%X AAm/BIS gel cassette synthesizedin water and aqueous solutions of tri(ethylene glycol) or poly(ethyleneglycol) 400

Migration ratios of SDS-PAGE Molecular Weight Standards (board range) in10%M 2%X acrylamide gel cassette synthesized in water or aqueoussolutions of tri(ethylene glycol) and poly(ethylene glycol) are shown inFIG. 3.

Example 23 Electrophoresis of 10%M 2%X AAm/BIS gel cassette synthesizedin water and aqueous solutions of tri(ethylene glycol)

Migration ratios of Kaleidoscope Prestained Standards in 10%M 2%Xacrylamide gel cassette synthesized in water and aqueous solutions oftri(ethylene glycol) are shown in FIG. 4.

Preparation of Methacrylamide Hydrogels

Example 24 Preparation of 10%M 2%Xmethacrylamide/N,N′-methylenebismethacrylamide hydrogels using aqueousglycerol as solvent

Aqueous solution of glycerol (75%) were prepared by mixing appropriateamount of water and glycerol methacrylamide (978.6 mg) andN,N′-methylenebismethacrylamide (21.4 mg) were added to the abovesolutions (9 g) in disposable glass vials. The monomer solution was thendegassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. The polymerizationwas then allowed to proceed at room temperature overnight under an argonenvironment to produce a hydrogel that was visually clear.

Opacity and reduction in mechanical integrity was observed when theabove methacrylamide hydrogel was equilibrated in water.

Preparation of 2-Hydroxyethyl Acrylate (HEA) Hydrogels

Example 25 Preparation of HEA/EGDA hydrogels using water as solvent

10%M HEA hydrogels at 3, 4, 5, 6, and 10%X were prepared by mixing theappropriate amount of HEA, EGDA. The monomer solution was then degassedby argon purging for 5 min prior to the addition of the initiator system(0.2 mol % initiator per double bond) composed of freshly made up 10%(w/v) APS and 10% (v/v) TEMED. The polymerization was then allowed toproceed at room temperature overnight under an argon environment.

All of the resultant polymers were not visually clear, the opacity wasobserved to increase with increasing %X.

Example 26 Preparation of 10%M 6.5%X HEA/EGDA hydrogels using aqueousethylene glycol as solvent

Aqueous solutions of ethylene glycol (20, 40, 60 and 80%) were preparedby varying amounts of ethylene glycol and water. HEA (951.5 mg) and EGDA(48.5 mg) were added to the above solutions (9 g) in disposable glassvials. The monomer solution was then degassed by argon purging for 5 minprior to the addition of the initiator system (0.2 mol % initiator perdouble bond) composed of freshly made up 10% (w/v) APS and 10% (v/v)TEMED. The polymerization was then allowed to proceed at roomtemperature overnight under an argon environment.

The polymers synthesized in 0 and 20% ethylene glycol solutions wereopaque. The polymer synthesized in 40% ethylene glycol solution wasslightly opalescence. The polymer synthesized in 60 and 80% ethyleneglycol solutions were visually clear and remained visually clear afterequilibration in water.

Example 27 Preparation of 10%M 6.5%X HEA/EGDA hydrogels using aqueoussolutions of poly(ethylene glycol) 200, tetrahydrofuran, or methanol assolvent

60% aqueous solutions of PEG 200, tetrahydrofuran, or methanol wereprepared. HEA (951.5 mg) and EGDA (48.5 mg) were added to the abovesolutions (9 g) in disposable glass vials. The monomer solution was thendegassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10%.(w/v) APS and 10% (v/v) TEMED. The polymerizationwas then allowed to proceed at room temperature overnight under an argonenvironment.

The polymers synthesized in 60% PEG 200, 60% tetrahydrofuran, and 60%methanol were visually clear. All gels were visually clear and remainedvisually clear after equilibration in water.

Preparation of 2-Hydroxyethyl Methacrylate (HEMA) Hydrogels

Example 28 Preparation of 5%X HEMA/EGDMA hydrogels using water assolvent

10%, 20%, 30% and 40%M HEMA hydrogels were prepared by mixing theappropriate amount of HEMA, EGDMA and water (10 g total) in disposableglass vials. The monomer solution was then degassed by argon purging for5 min prior to the addition of the initiator system (0.2 mol % initiatorper double bond) composed of freshly made up 10% (w/v) APS and 10% (v/v)TEMED. The polymerization was then allowed to proceed at roomtemperature overnight under an argon environment.

All of the resultant polymers were highly opaque and had littlemechanical strength.

Example 29 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous ethylene glycol, tri(ethylene glycol), PEG400 or PEG 6,000 as solvent

Aqueous solutions of ethylene glycol, tri(ethylene glycol), PEG 400 orPEG 6,000 (40, 45, 50, 60 and 70%) were prepared. HEMA (1.442 g) andEGDMA (57.8 mg) were added to the above solutions (8.5 g) in disposableglass vials. The monomer solution was then degassed by argon purging for5 min prior to the addition of the initiator system (0.2 mol % initiatorper double bond) composed of freshly made up 10% (w/v) APS and 10% (v/v)TEMED. Two 375 μl samples were pipetted into disposable cuvettes(10×10×45 mm³) and the polymerization was then allowed to proceed atroom temperature overnight under an argon environment.

Example 30 Preparation of 15%M HEMA/EGDMA hydrogels for turbiditymeasurements using 50% PEG 200 as solvent

Aqueous solution of PEG 200 (50%) was prepared by mixing the appropriateamount of PEG 200 and water. 15%M HEMA hydrogels with 0, 2.5, 5, 7.5 and10%X were prepared by mixing the appropriate amounts of HEMA, EGDMA and50% PEG solution (10 g total) in disposable glass vials. The monomersolution was then degassed by argon purging for 5 min prior to theaddition of the initiator system (0.2 mol % initiator per double bond)composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375μl samples were pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 31 Preparation of 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using 50% PEG 200 as solvent

Aqueous solution of PEG 200 (50%) was prepared by mixing the appropriateamount of PEG 200 and water. 5%X HEMA hydrogels with 7.5, 10, 12.5, 15,20, 40%T were prepared by mixing the appropriate amounts of HEMA, EGDMAand 50% PEC solution (10 g total) in disposable glass vials. The monomersolution was then degassed by argon purging for 5 min prior to theaddition of the initiator system (0.2 mol % initiator per double bond)composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375μl samples were pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 32 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous propylene glycol, tri(propylene glycol) orPPG 425 as solvent

Aqueous solutions of propylene glycol, tri(propylene glycol) or PPG 425(30, 35, 40, 45 and 50%) were prepared. HEMA (1.442 g) and EGDMA (57.8mg) were added to the above solutions (8.5 g) in disposable glass vials.The monomer solution was then degassed by argon purging for 5 min priorto the addition of the initiator system (0.2 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two375 μl samples were pipetted into disposable cuvettes (10×10×45 mm³) andthe polymerization was then allowed to proceed at room temperatureovernight under an argon environment.

Example 33 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous PEG dimethyl ether 500 as solvent

Aqueous solutions of PEG dimethyl ether 500 (30, 35, 40, 45 and 50%)were prepared. HEMA (1.442 g) and EGDMA(57.8 mg) were added to the abovesolutions (8.5 g) in disposable glass vials. The monomer solution wasthen degassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 34 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous ethylene glycol monomethyl ether, ethyleneglycol monoethyl ether or ethylene glycol monobutyl ether as solvent

Aqueous solutions of ethylene glycol monomethyl ether, ethylene glycolmonoethyl ether or ethylene glycol monobutyl ether (30, 35, 40, 45 and50%) were prepared. HEMA (1.442 g) and EGDMA (57.8 mg) were added to theabove solutions (8.5 g) in disposable glass vials. The monomer solutionwas then degassed by argon purging for 5 min prior to the addition ofthe initiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 35 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous poly(ethylene glycol-co-propylene glycol)2,500 (poly(eg-co-pg) 2,000), poly(ethylene glycol-co-propylene glycol)12,000 (poly(eg-co-pg) 12,000) or poly(ethylene glycol-block-propyleneglycol-block-ethylene glycol) 1,900 (poly(eg-b-pg-eg) 1,900) as solvent

Aqueous solutions of poly(eg-co-pg) 2,000, poly(eg-co-pg) 12,000 orpoly(eg-b-pg-eg) 1,900 (30, 35, 40, 45 and 50%) were prepared. HEMA(1.442 g) and EGDMA (57.8 mg) were added to the above solutions (8.5 g)in disposable glass vials. The monomer solution was then degassed byargon purging for 5 min prior to the addition of the initiator system(0.2 mol % initiator per double bond) composed of freshly made up 10%(w/v) APS and 10% (v/v) TEMED. Two 375 μl samples were pipetted intodisposable cuvettes (10×10×45 mm³) and the polymerization was thenallowed to proceed at room temperature overnight under an argonenvironment.

Example 36 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous PEG 400 or PPG 425 as solvent

Aqueous solutions of PEG 400 or PPG 425 (30, 50, 70 and 90%) wereprepared. HEMA (1.442 g) and EGDMA (57.8 mg) were added to the abovesolutions (8.59) in disposable glass vials. The monomer solution wasthen degassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 37 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous solutions of poly(eg-b-pg-b-eg) 1900 and PEG400 mixtures as solvent

40% aqueous solutions of poly(eg-b-pg-b-eg) 1900 and PEG 400 mixtures(0, 12.5, 25, 50, 75, 87.5 and 100% poly(eg-b-pg-b-eg) 1900) wereprepared. HEMA (1.442 g) and EGDMA (57.8 mg) were added to the abovesolutions (8.5 g) in disposable glass vials. The monomer solution wasthen degassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 38 Preparation of 15%M 5%X HEMA/EGDMA hydrogels for turbiditymeasurements using aqueous solutions of ethylene glycol monomethyl etherand PEG 200 mixtures as solvent

35% aqueous solutions of ethylene glycol monomethyl ether and PEG 200mixtures (0, 14, 28, 57, 86 and 100% ethylene glycol monomethyl ether)were prepared. HEMA (1.442 g) and EGDMA (57.8 mg) were added to theabove solutions (8.5 g) in disposable glass vials. The monomer solutionwas, then degassed by argon purging for 5 min prior to the addition ofthe initiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 39 Preparation of 5%X HEMA/EGDMA hydrogels for swelling testsusing aqueous tri(ethylene glycol) as solvent

Aqueous solution of tri(ethylene glycol) (60%) were prepared. 20, 40, 60and 80%M HEMA/EGDMA hydrogels were prepared by mixing the appropriateamount of HEMA, EGDMA and the above 60% tri(ethylene glycol) solution indisposable glass vials (10 g total). The monomer solution was thendegassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. The polymerizationwas then allowed to proceed at room temperature overnight under an argonenvironment.

Example 40 Preparation of 10%M 5%X HEMA/EGDMA hydrogels for swellingtests using water and aqueous solutions of PEG 200 or PEG 4000 assolvent

Aqueous solutions of PEG 200 or PEG 4000 (50%) were prepared. HEMA(1.442 g) and EGDMA (57.8 mg) were added to the above solutions (8.5 g)in disposable glass vials. The monomer solution was then degassed byargon purging for 5 min prior to the addition of the initiator system(0.2 mol % initiator per double bond) composed of freshly made up 10%(w/v) APS and 10% (v/v) TEMED. The polymerization was then allowed toproceed at room temperature overnight under an argon environment.

Example 41 Preparation of 15%M 4%X HEMA/EGDMA membrane forelectrophoretic separation analysis using aqueous solutions of PEG 200as solvent

Unwoven poly(ethyleneterephthalate) (PET) sheets that served as amechanical support were treated with aqueous solution of Teric BL8 (0.5%(v/v)), Huntsman Corp. Australia) a non-ionic surfactant used to improvesurface wettability.

Aqueous solution of PEG 200 (80%) were prepared. 15%M 4%X HEMA/EGDMAmixtures with the above PEG 200 solution were polymerized into thinmembranes with Teric BL8 treated unwoven PET sheet as the supportingsubstrate.

Evaluation of HEMA Hydrogels

Turbidity testing

All HEMA hydrogels which were visually clear after the synthesisremained visually clear after the solvent was exchanged with water.

Example 42 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous ethylene glycol, tri(ethylene glycol), PEG 400 or PEG 6,000solutions at 500 nm

Turbidity results of polymers synthesized according to Example 29 areshown in FIG. 5.

Example 43 Turbidity of 15%M HEMA/EGDMA hydrogels synthesized in 50%aqueous PEG 200 solution at 500 nm

Turbidity results of polymers synthesized according to Example 30 areshown in FIG. 6.

Example 44 Turbidity of 5%X HEMA/EGDMA hydrogels synthesized in 50%aqueous PEG 200 solution at 500 nm

Turbidity results of polymers synthesized according to Example 31 areshown in FIG. 7.

Example 45 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous propylene glycol, tri(propylene glycol) or PPG 425 as solvent

Turbidity results of polymers synthesized according to Example 32 areshown in FIG. 8.

Example 46 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous PEG dimethyl ether 500 solutions

Turbidity results of polymers synthesized according to Example 33 areshown in FIG. 9.

Example 47 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous ethylene glycol monomethyl ether, ethylene glycol monoethylether or ethylene glycol monobutyl ether as solvent

Turbidity results of polymers synthesized according to Example 34 areshown in FIG. 10.

Example 48 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous poly(ethylene glycol-co-propylene glycol) 2,500 (poly(eg-co-pg)2,000), poly(ethylene glycol-co-propylene glycol) 12,000 (poly(eg-co-pg)12,000) or poly(ethylene glycol-block-propylene glycol-block-ethyleneglycol) 1,900 (poly(eg-b-pg-eg) 1,900) as solvent

Turbidity results of polymers synthesized according to Example 35 areshown in FIG. 11.

Example 49 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous PEG 400 or PPG 425 as solvent

Turbidity results of polymers synthesized according to Example 36 areshown in FIG. 12.

Example 50 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous solutions of poly(eg-b-pg-b-eg) 1900 and PEG 400 mixtures assolvent

Turbidity results of polymers synthesized according to example 37 areshown in FIG. 13.

Example 51 Turbidity of 15%M 5%X HEMA/EGDMA hydrogels synthesized inaqueous solutions of ethylene glycol monomethyl ether and PEG 200mixtures as solvent

Turbidity results of polymers synthesized according to Example 38 areshown in FIG. 14.

Example 52 Swelling test (water) of 5%X HEMA/EGDMA hydrogels at 20, 40,60, 80%M synthesized in 60% aqueous tri(ethylene glycol) solution

Hydrogel ESC (water) 20% M 5% X 0.81 40% M 5% X 0.72 60% M 5% X 0.56 80%M 5% X 0.54

Example 53 Swelling test (water) of 15%M 5%X HEMA/EGDMA hydrogelssynthesized in 50% aqueous solutions of PEG 200 or PEG 4000

ESC(water) for 15%M 5%X HEMA/EGDMA hydrogel synthesized in 50% PEG 200solution was found to be 0.65.

ESC(water) for 15%M 5%X HEMA/EGDMA hydrogel synthesized in 50% PEG 4000solution was found to be 0.83.

Example 54 Swelling test (40% aqueous solutions of ethylene glycol, PEG600, PEG 4000 or PEG 6000) of 15%M 5%X HEMA/EGDMA hydrogels synthesizedin 50% aqueous solutions of PEG 200 or PEG 400

Hydrogels prepared in Example 40 were immersed in water (500 g) for 1week during which the immersing solution (water) was exchanged on adaily basis. The gel was then dried in a 40° C. oven for 1 week. Thedried gels were then immersed in 50% aqueous solutions of ethyleneglycol, PEG 600, PEG 4000 or PEG 6000) for 1 week during which theimmersing solution was exchanged on a daily basis. The ESC of the gelsare shown in the following table. ESC ESC ESC ESC (40% PEG (40% PEG (40%PEG (40% EG) 600) 4000) 6000) 15% M 5% X hydrogels 0.98 2.99 1.31 1.14synthesized in 50% PEG 200 15% M 5% X hydrogels 1.30 3.48 3.00 2.45synthesized in 50% PEG 4000

Example 55 Electrophoresis separation analysis of 15%M 4%X HEMA/EGDMAmembrane synthesized in 80% aqueous PEG 200 solution

Samples of known molecular weight and size were run through a Gradiflow™BF 200 unit to investigate the relative pore size formed in HEMAhydrogel networks. The protein standards were placed in a buffersolution and run by current from the stream 1 section of the unit abovethe membrane. Proteins smaller than the pores of the membrane will passthrough the membrane into the stream 2 section of the unit. The largerproteins will be recycled back into the stream 1 section. Ten μl samplesfrom both the two streams of the unit are taken every 10 minutes anddetected using SDS-PAGE. The migration pattern should indicate whatsized samples passed through the membrane. More details on theconstruction and operation of this unit can be found in U.S. Pat. No.5,650,055, U.S. Pat. No. 5,039,386, WO 00/56792 and WO 00/13776,incorporated herein by reference.

The separation and migration pattern of Bovine serum albumin (MW 67,000)by a 15%M 4%X HEMA/EGDMA membrane synthesized in 80% aqueous PEG 200solution (Example 41) using 40 mM MES bis-TRIS buffer is shown in FIG.15;

Preparation of Poly(ethylene glycol) Methacrylate (HEMA) Hydrogels

Example 56 Preparation of 15%M 5%X PEGMA 526/EGDMA hydrogels forturbidity measurements using aqueous PEG 400 or PPG 425 as solvent

Aqueous solutions of PEG 400 or PPG 425 (0, 30, 50 and 70%) wereprepared. PEGMA (1.485 g) and EGDMA (14.7 mg) were added to the abovesolutions (8.5 g) in disposable glass vials. The monomer solution wasthen degassed by argon purging for 5 min prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. Two 375 μl sampleswere pipetted into disposable cuvettes (10×10×45 mm³) and thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 57 Turbidity of 15%M 5%X PEGMA 526/EGDMA hydrogels synthesizedin aqueous PEG 400 or PPG 425 as solvent

Turbidity results of polymers synthesized according to Example 56 areshown in FIG. 16.

Preparation of optically clear hydrogels

Example 58 ¹³C NMR relaxation measurements of acrylamide hydrogels

Monomer solutions (2 g) were prepared by dissolving AAm and Bis in theappropriate amount of D₂O (10% TMSPA-Na, 0.2 g), water and PEG-400. Themonomer solution was then degassed by argon purging prior to theaddition of the initiator system composed of freshly made up 10% (w/v)APS and 10% (v/v) TEMED (0.05 mol % initiator per double bond). Thismixture was immediately pipetted into 5 mm NMR tube (0.38 mm wallthickness) and the polymerization was allowed to proceed at roomtemperature overnight under an argon environment.

¹³C NMR spectra were obtained using a Varian Unity Plus 400 spectrometeroperating at 100 MHz. Spin-lattice relaxation times (T₁) were measuredby the inversion-recovery method at 25° C. Recycled delays were set to7s (>3T₁), with delay times (τ) of 10, 50, 100, 200, 300, 400, 500, 600,700, 800, and 1000 ms. The T₁ parameters were calculated by fitting thedata to the following equation.l(τ)=l(τ=0) (1−2 exp(−τ/T ₁))  (4)when l is the intensity of the transformed peaks.

Example 59 Real-time viscosity measurements of acrylamidepolymerizations

Monomer solutions (200 g) were prepared by dissolving AAm and Bis in theappropriate amount of water and PEG-400. The monomer solution was thendegassed by argon purging prior to the addition of the initiator systemcomposed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED (0.05 mol% initiator per double bond). The viscosity of the reaction mixture wasmeasured by a Brookfield® DV-II+ viscometer (0.3 rpm, LV-3 spindle). Theexperiments were performed in a glove box with controlled oxygen levels(<0.1% O₂).

Viscosity measurements of the polymerizations are shown in FIG. 18.Times at which phase separation was observed in the samples arerepresented by dark coloured points (circle).

Example 60 Preparation of acrylamide hydrogels for swelling studies

Monomer solution (10 g) was prepared by dissolving AAm and Bis in anappropriate amount of water and PEG-400 in disposable glass vials. Themonomer solution was then degassed by argon purging prior to theaddition of the initiator system (0.2 mol % initiator per double bond),composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

Example 61 Kinetic swelling studies of acrylamide hydrogels

The gel made according to the above procedure was immersed in water (500g) for 1 week during which the immersing solution (water) was exchangedon a daily basis. The gel was then dried in a 40° C. oven for 1 week andre-swelled in water. The weight of the swollen gel was continuouslymonitored for 48 hours.ESC of the gel was determined by the following equation:${{Equilibrium}\quad{solvent}\quad{{content}({ESC})}} = \frac{{{weight}( {{swollen}\quad{gel}} )} - {{weight}( {{dried}\quad{gel}} )}}{{weight}( {{dried}\quad{gel}} )}$

Example 62 Preparation of 15%M 5%X HEMA/EGDMA hydrogels using aqueousethylene glycol monomethyl ether as solvent

Aqueous solutions of ethylene glycol monomethyl ether (80, 85 and 90%)were prepared. HEMA (1.442 g) and EGDMA (57.8 mg) were added to theabove solutions (8.5 g) in disposable glass vials. The monomer solutionwas then degassed by argon purging prior to the addition of theinitiator system (0.2 mol % initiator per double bond) composed offreshly made up 10% (w/v) APS and 10% (v/v) TEMED. The polymerizationwas then allowed to proceed at room temperature overnight under an argonenvironment. All resultant gels were visually clear.

Example 63 ¹³C T₁ (25° C., 100 MHz) for 20%M 2%X acrylamide hydrogelssynthesized in the presene of various amount of PEG-400

% PEG-400 T₁ (α-carbon) T₁ (β-carbon) T₁ (carbonyl) 2.5 240 125 1330 7.5240 135 1350 12.5 261 140 1400 17.5 270 155 1400 22.5 340 180 1730 27.5420 230 2185

Example 64 ESC (water) of AAm/BIS hydrogels from kinetic swellingstudies

% PEG-400 Time(hr) 7.5 12.5 17.5 22.5 27.5 0.5 3.14 3.34 3.34 2.99 2.701 3.52 3.82 3.81 3.34 2.99 1.5 3.81 4.14 4.18 3.59 3.23 2 4.05 4.44 4.483.81 3.42 3 4.47 4.97 5.10 4.20 3.76 4 4.86 5.55 5.50 4.54 4.05 5 5.236.03 6.04 4.93 4.42 24 12.04 13.16 13.02 9.3 6.84 48 15.22 16.40 16.5313.21 8.58

Example 65 Preparation of 20%M 2%X AAm/Bis hydrogels using aqueousPEG-400 as solvent

Monomer solutions (10 g) were prepared by dissolving AAm and Bis in theappropriate amount of water and PEG 400 in disposable glass vials. Themonomer solution was then degassed by argon purging prior to addition ofthe initiator system (0.05 mol % initiator per double bond) composed offreshly made up 10% (v/v) TEMED and 10% (w/v) APS. The polymerizationwas then allowed to proceed at room temperature overnight under an argonenvironment.

Example 66 Optical properties of 20%M 2%X AAm/Bis hydrogels synthesizedusing aqueous PEG-400 as solvent

Turbidity results and images of polymers synthesized according toExample 65 are shown in FIG. 19.

Example 67 Preparation of optically clear HEMA/EGDMA hydrogels usingaqueous propylene glycol as solvent

HEMA hydrogels (10%, 20%, 30%, 40%, 50%, 60%M) were prepared by mixingthe appropriate amount of HEMA, EGDMA (1%X, 2%X, 4%X, 6%X, 8%X),propylene glycol and water (10 g total) in disposable glass vials. Themonomer solution was then degassed by argon purging for 5 min prior tothe addition of the initiator system (0.1 mol % initiator per doublebond) composed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED. Thepolymerization was then allowed to proceed at room temperature overnightunder an argon environment.

The propylene glycol content of each reaction mixture was varied in 2.5%(increments from 0%) until an optically clear hydrogel is obtained.

Example 68 Critical propylene glycol concentrations for the formation ofvisually clear HEMA hydrogels at various %M and %X

FIG. 20 shows the critical propylene glycol concentrations for theformation of visually hydrogels at various %M and %X.

Example 69 Real-time viscosity measurements of 20%M 2%X HEMApolymerizations using aqueous propylene glycol as solvent

Monomer solutions (200 g) were prepared by mixing HEMA and EGDMA in theappropriate amount of water and PG. The monomer solution was thendegassed by argon purging prior to addition of the initiator systemcomposed of freshly made up 10% (w/v) APS and 10% (v/v) TEMED (0.1 mol %initiator per double bond). The viscosity of the reaction mixture wasmeasured by a Brookfield® DV-II+ viscometer (0.3 rpm, LV-3 spindle). Theexperiments were performed in a glove box with controlled oxygen levels(<0.1% O₂).

Viscosity measurements of the polymerizations are shown in FIG. 21.Times at which phase separation was observed in the samples arerepresented by dark coloured points (circle).

Scanning electron microscopy (SEM)

SEM analysis was performed on the hydrogels synthesized in Examples 3and 4.

After equilibration in water, a piece of hydrogel (5×5 mm) was mountedvertically onto a SEM stub and cryogenically fractured in liquidnitrogen. The water from the fractured surface of the gel was sublimedat 60° C. for 60 min. The gel was then cooled to −190° C. and images ofthe fractured polymer were taken at 10,000× magnification using a XL30field emission scanning electron microscope (FESEM).

Example 70 SEM analysis of 10%M 2%X AAm/BIS hydrogels synthesized usingwater, 50% ethylene glycol, or 50% propylene glycol as solvent

SEM images of the polymers synthesized according to Example 3 and 4 areshown in FIG. 22.

SUMMARY

Examples 2 to 23 show that the following:

Acrylamide hydrogels can undergo polymerization-induced phase separationwhen it is synthesized in solvents containing poly(ethylene glycol) with3 repeating units or more.

Turbidity testing showed that the onset of opacity (i.e. phaseseparation) occurs at lower concentrations of poly(ethylene glycol) withincreasing molecular weight of poly(ethylene glycol).

Acrylamide hydrogels synthesized in the presence of water-solubleentities have in general, larger pores than those synthesized in water.Such gels however cannot be synthesized in solvents containing highconcentrations of poly(ethylene glycol) with high molecular weight.

It is well known that when methacrylamide is polymerized in water, anopaque polymer mass is obtained. Example 24 showed that visually clearhydrogels can be obtained from methacrylamide by using hydro-organicsolution as the polymerization solvent. Such hydrogels, however, becameopaque and lost their mechanical integrity when the organic solvent wassubsequently exchanged with water. This demonstrated that although byusing a hydro-organic solution as the polymerization solvent, a visuallyclear hydrogel can be obtained from monomers that producewater-immiscible polymers, many of the resultant hydrogels cannot beused in aqueous media.

Examples 25-27 show that:

HEA hydrogels that are synthesized using water as solvent are opaque andhave poor mechanical integrity.

Visually clear HEA hydrogels can be synthesized by careful selection ofwater-miscible entities. Such gels remained visually clear after thewater-miscible entities were exchanged with water. This contrasts withthe teaching from prior art observations made in methacrylamidehydrogels.

Examples 28-37 and 42-51 show that:

HEMA hydrogels that are synthesized in water are opaque and have poormechanical integrity.

Visually clear HEMA hydrogels can be synthesized by careful selection ofwater-miscible entities. Such gels remained visually clear after thewater-miscible entities were exchanged with water.

HEMA hydrogels have very different behaviour to acrylamide hydrogels.Polymerization-induced phase separation occurs at low concentrations ofwater-miscible entities (e.g. poly(ethylene glycol)), and the gelsbecome more visually clear and the mechanical properties of such gelsincreases when the concentrations of water-miscible entities increases.This contrasts with prior art acrylamide hydrogels, which state thathigh concentrations of water-miscible entities would lead to phaseseparations.

Unexpectedly, turbidity testing shows that in contrast to acrylamidehydrogels, poly(ethylene glycol) with higher molecular weight improvesthe visual and mechanical properties of the resultant gel (FIG. 5). Thiscontrasts with prior art acrylamide systems, which state thatwater-miscible entities with high molecular weight would lead to phaseseparation.

FIG. 7 (Example 31 and 44) shows that visually clear HEMA hydrogels canbe obtained from reaction mixtures with low initial monomerconcentrations. This contrasts with prior art HEMA gels.

FIGS. 8 and 12 (Example 32, 36 and 45, 49) demonstrate the usage ofpoly(propylene glycol) as water-miscible entities. The usage ofpoly(propylene glycol) has not been reported in the literature onhydrogel synthesis.

FIGS. 9 and 10 (Example 33-34 and 46-47) demonstrate the usage ofpoly(ethylene glycol) derivatives (i.e. alkyl ether) as water-miscibleentities. The usage of such derivatives has not been reported in theliterature on acrylamide hydrogel synthesis.

FIG. 11 (Example 35 and 48) demonstrate the usage of random and blockcopolymers of poly(ethylene glycol) and poly(propylene glycol) aswater-miscible entities. The usage of such water-miscible entities hasnot been reported previously.

FIGS. 13 and 14 (Example 36-37 and 49-51) demonstrate the usage of twodifferent types of water-miscible entities together in the same solventsystem. The usage of such mixtures of water-miscible entities have notbeen reported previously.

Example 52 shows that by careful selection of the water-miscibleentities, HEMA hydrogels with high water swelling properties can beformed from monomer mixtures with low monomer concentrations (i.e.<50%M). It also shows the increase in water swelling properties withdecreasing total monomer concentrations. This contrasts with the priorwhich states the opposite.

Examples 52 and 53 show that water swelling properties of HEMA hydrogelsare dependent upon the initial monomer concentration, the types ofwater-miscible entities and the concentration of water-miscibleentities.

Example 53 further demonstrates that the water swelling properties ofthe hydrogels increases when the molecular weight of the water-miscibleentities (i.e. poly(ethylene glycol) is increased.

Example 54 shows the swelling properties of two different hydrogels.Hydrogel A was synthesized in the presence of a water-miscible entitywith low molecular weight; hydrogel B was synthesized in the presence ofa water miscible entity with high molecular weight.

Swelling of Hydrogel A and B in mixtures composed of water and organicsolvents with different molecular weight shows that:

Hydrogel B swells more in all solvents.

Hydrogel A has low swelling properties in solvents with organic solventswith high molecular weight.

Hydrogel B has significantly higher swellings in solvents with highmolecular weight than Hydrogel A.

The above observations show that as the molecular weight of thewater-miscible entities increases, the pore size of the gels becomedependent upon the size of the water-miscible entities. Such gels havemacroporous pores and hence are able to swell more in solvents with highmolecular weight solutes, because of the increased diffusion of organicsolvent with high molecular weights into the gel.

Examples 56 and 57 demonstrate the usages of poly(ethylene glycol) andpoly(propylene glycol) as water-miscible entities in other hydrogelsprepared from α,ω-(meth)acryloyloxy monomers. Poly(ethylene glycol)methacrylate was used in these examples. The present invention extendsto derivatives of HEMA and HEA, that is, monomers with the same(meth)acrylate ester structure with HEMA and HEA, but different sidechains.

Example 58 and 63 show that PIPS occur in 20%M 2%X acrylamide hydrogelssynthesized in the presence of 22.5 and 27.5% PEG-400, but can beavoided by the careful selection of the polymerization solvent. It istherefore possible to prepare visually clear hydrogels even when thepolymerization solvent is immiscible with the corresponding linearpolymer analogues.

FIG. 17 is a schematic diagram of the formation process of 20%Macrylamide hydrogel, it demonstrates the relationship between the‘freezing concentration’ of the reaction mixture, the phase boundary,and the concentration and properties of the water-miscible entity whichalter the region of immiscibility on the diagram.

Example 59 and FIG. 18 demonstrate the relationship between the‘freezing concentration’ of the reaction mixture and the phase boundary,it can be seen that visually clear gels can be obtained. In systemswhere the ‘freezing concentration’ of the reaction mixtures is reachedbefore the onset of PIPS.

Examples 60, 61, and 64 show that hydrogels prepared by the approach ofthis invention have superior swelling properties to that prepared bysystems that reaches the phase boundary before the gel point (22.5 and27.5% PEG-400).

Example 62 shows that by using a mixture of water and water-miscibleentities as the polymerization solvent, visually clear HEMA hydrogelscan be prepared even when the polymerization solvent is immiscible withthe corresponding linear polymer analogues which are water immiscible.

Examples 65 and 66 show that hydrogels with very different opticalproperties can be obtained by controlling the ‘freezing point’ of thereaction mixture.

Examples 67 and 68 show that visually clear HEMA hydrogels, at differenttotal monomer concentration and crosslinker concentration, can besynthesized by careful selection of water miscible entities. Such gelsremained visually clear after the water-miscible entities were exchangedwith water. The critical propylene glycol concentration (and hencecritical water content of the reaction mixture) required to obtain aclear gel in these systems are shown in FIG. 20. It can be seen fromFIG. 20 that in contrast to the reported values of around 50%, themaximum water content of the reaction mixtures to produce a clearhydrogel is dependent upon both %M and %X. For example, the maximumwater content is 30% at 60%M 8%X, and 50% at 10%M 1%X.

Example 69 and FIG. 21 demonstrate the relationship between the‘freezing concentration’ of the reaction mixture and the phase boundary;it can be seen that visually clear gels can be obtained. In systemswhere the ‘freezing concentration’ of the reaction mixtures is reachedbefore the onset of PIPS.

Example 70 shows that when compared with Mm hydrogels obtained byexisting methods (water as polymerization solvent), hydrogels preparedby the approach of this invention have significantly different pore sizeand pore size distribution.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A process for producing a polymeric hydrogel having a networkcontaining macropores and micropores comprising: (a) forming a mixtureby adding at least one monomer having at least one double bond, at leastone crosslinker having at least two double bonds, an initiation system,and an organic additive to form a hydro-organic system with water; and(b) allowing the monomer and crosslinker to copolymerize to form ahydrogel having a polymeric network containing macropores andmicropores.
 2. The process according to claim 1 wherein the monomerhaving at least one double bond is selected from the group consisting ofpolyol esters of acrylic acid, polyol esters of methacrylic acid, andmixtures thereof.
 3. The process according to claim 1 wherein themonomer having at least one double bond is one or more hydrophilicmonomers of polyol esters of acrylic or methacrylic acid.
 4. The processaccording to claim 1 wherein the polyol is selected from the groupconsisting of polyethylene glycol, polyethylene glycol esters or ethers,polypropylene glycol, polypropylene glycol esters or ethers, random orblock copolymers of ethylene glycol and propylene glycol, glycerol,pentaerythritol, ethylene glycol, propylene glycol, and mixturesthereof.
 5. The process according to claim 1 wherein the monomer ishydroxyethyl methacrylate (HEMA).
 6. The process according to claim 1wherein the monomer is used at a concentration from 5 to 50%.
 7. Theprocess according to claim 1 wherein the crosslinker having at least twodouble bond is selected from the group consisting of esters of acrylicand/or methacrylic acid, acrylic or methacrylic acid with variouspolyols, and mixtures thereof.
 8. The process according to claim 7wherein the polyol is selected from the group consisting of polyethyleneglycol, polypropylene glycol, random or block copolymers of ethyleneglycol and propylene glycol, glycerol, pentaerythritol, ethylene glycol,propylene glycol, and mixtures thereof.
 9. The process according toclaim 1 wherein the crosslinker is ethylene glycol dimethacrylate(EGDMA).
 10. The process according to claim 1 wherein the crosslinker isused at a concentration of greater than about 50% in the mixture ofcrosslinkers; more preferably greater than about 80%.
 11. The processaccording to claim 1 wherein the polymeric hydrogel is made from amixture of monomer content of about 10 to 40%M and crosslinker of about1 to 30%X before polymerization.
 12. The process according to claim 11,utilizing HEMA with EGDMA wherein compositions of monomer mixture ofHEMA with EGDMA are less than about 40% M and less than about 20% X. 13.The process according to claim 1 wherein a free radical producing methodis used as an initiation system.
 14. The process according to claim 13wherein the initiation system is formed by redox, thermal or photoinitiators.
 15. The process according to claim 14 wherein the redoxinitiator is formed by ammonium persulphate (APS) withN,N,N′,N′-tetramethylethylenediamine (TEMED).
 16. The process accordingto claim 1 wherein the organic additive is a hydrophilic polymermiscible with water and miscible with a linear polymer produced from themonomer used for copolymerization, or a hydrophilic polymer misciblewith water and having a similar solubility parameter (±10(MPa)^(0.5)) tothat of a polymer produced from the monomer used for copolymerization.17. The process according to claim 16 wherein the organic additive issingle entity acting as both a porogen to form macropores during thepolymerization and a solvent with water to form the hydro-organicsolvent.
 18. The process according to claim 17 wherein the organicadditive is selected from the group consisting of ethylene glycol,polyethylene glycol, propylene glycol, polypropylene glycol, random orblock copolymers of ethylene glycol, random or block copolymers ofpolyethylene glycol, random or block copolymers of propylene glycol,random or block copolymers of polypropylene glycol, ethylene glycolhaving an ester or ether end group, polyethylene glycol having an esteror ether end group, propylene glycol having an ester or ether end group,polypropylene glycol having an ester or ether end group, and mixturesthereof.
 19. The process according to claim 18 wherein the organicadditive has the following general formulation:

Rd 1, R₄═H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), —C(═O)—R₅ (R₅═(CH₂)_(x)—CH₃(x=0-4)) R₂, R₃═H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), OH
 20. The processaccording to claim 19 wherein the organic additive is a polyethyleneglycol or polypropylene glycol.
 21. The process according to claim 20wherein the polyethylene glycol has a molecular weight range from about100 to about 100,000.
 22. The process according to claim 20 wherein thepolypropylene glycol has a molecular weight range from about 100 toabout 100,000.
 23. The process according to claim 16 wherein the organicadditive is a copolymer with a hydrophilic component and a hydrophobiccomponent.
 24. The process according to claim 23 wherein the organicadditive is a copolymer of polyethylene glycol with polypropyleneglycol.
 25. A polymeric hydrogel having a network containing macroporesand micropores produced by the process according to claim
 1. 26. Apolymeric hydrogel comprising a network of macropores and microporesformed by copolymerizing at least one monomer having at least one doublebond and at least one crosslinker having at least two double bonds inthe presence of an organic additive forming a hydro-organic system withwater.
 27. The hydrogel according to claim 26 wherein the monomer havingat least one double bond is selected from the group consisting of polyolesters of acrylic acid, polyol esters of methacrylic acid, and mixturesthereof.
 28. The hydrogel according to claim 26 wherein the monomerhaving at least one double bond is one or more hydrophilic monomers fromthe polyol esters of acrylic or methacrylic acid.
 29. The hydrogelaccording to claim 27 wherein the polyol is selected from the groupconsisting of polyethylene glycol, polyethylene glycol esters or ethers,polypropylene glycol, polypropylene glycol esters or ethers, random orblock copolymers of ethylene glycol and propylene glycol, glycerol,pentaerythritol, ethylene glycol, propylene glycol, and mixturesthereof.
 30. The hydrogel according to claim 26 wherein the monomer ishydroxyethyl methacrylate (HEMA).
 31. The hydrogel according to claim 26wherein the monomer is used at a concentration from 5 to 50%.
 32. Thehydrogel according to claim 26 wherein the crosslinker having at leasttwo double bonds is selected form the group consisting of esters ofacrylic and/or methacrylic acid, acrylic or methacrylic acid withvarious polyols, and mixtures thereof.
 33. The hydrogel according toclaim 32 wherein the polyol is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, random or block copolymers ofethylene glycol and propylene glycol, glycerol, pentaerythritol, andethylene glycol, propylene glycol which are fully or partly esterified,and mixtures thereof.
 34. The hydrogel according to claim 26 wherein thecrosslinker is ethylene glycol dimethacrylate (EGDMA).
 35. The hydrogelaccording to claim 26 wherein the crosslinker is used at greater thanabout 50% in the mixture of crosslinkers; more preferably greater thanabout 80%.
 36. The hydrogel according to claim 26 wherein the polymerichydrogel is made from a mixture of monomer content of 10 to 40%M andcrosslinker of 1 to 30%X before polymerization.
 37. The hydrogelaccording to claim 36, wherein compositions of monomer mixture of HEMAwith EGDMA are less than about 40% M and less than about 20% X.
 38. Thehydrogel according to claim 26 wherein a free radical producing methodis used as initiation system.
 39. The hydrogel according to claim 38wherein the initiation system is formed by redox, thermal or photoinitiators.
 40. The hydrogel according to claim 39 wherein the redoxinitiator is formed by ammonium persulphate (APS) withN,N,N′,N′-tetramethylethylenediamine (TEMED).
 41. The hydrogel accordingto claim 26 wherein the organic additive is a hydrophilic polymermiscible with water and miscible with a linear polymer produced from themonomer used for copolymerization; or a hydrophilic polymer misciblewith water and has a similar solubility parameter (±10(MPa)^(0.5)) tothat of a polymer produced from the monomer used for copolymerization.42. The hydrogel according to claim 41 wherein the organic additive issingle entity acting as both a porogen to form macropores during thepolymerization and a solvent with water to form the hydro-organicsolvent.
 43. The hydrogel according to claim 42 wherein the organicadditive is selected from the group consisting of ethylene glycol,polyethylene glycol, propylene glycol, polypropylene glycol, random orblock copolymers of ethylene glycol, random or block copolymers ofpolyethylene glycol, random or block copolymers of propylene glycol,random or block copolymers of polypropylene glycol, ethylene glycolhaving an ester or ether end group, polyethylene glycol having an esteror ether end group, propylene glycol having an ester or ether end group,polypropylene glycol having an ester or ether end group, and mixturesthereof.
 44. The hydrogel according to claim 43 wherein the organicadditive has the following general formulation:

R₁, R₄=H, CH₃, —(CH₂)_(x)—CH₃ (x=1-4), —C(═O)—R₅ (R₅═(CH₂)_(x)—CH₃(x=0-4)) R₂, R₃=H, CH₃, —(CH₂)_(x)—CH₃ (x=14), OH
 45. The hydrogelaccording to claim 44 wherein the organic additive is a polyethyleneglycol or polypropylene glycol.
 46. The hydrogel according to claim 45wherein the polyethylene glycol has a molecular weight range from about100 to about 100,000.
 47. The hydrogel according to claim 45 wherein thepolypropylene glycol has a molecular weight range from about 100 toabout 100,000.
 48. The hydrogel according to claim 41 wherein theorganic additive is a copolymer with a hydrophilic component and ahydrophobic component.
 49. The hydrogel according to claim 48 whereinthe organic additive is a copolymer of polyethylene glycol withpolypropylene glycol.
 50. The hydrogel according to claim 26 beingvisually clear.
 51. A separation medium formed from the polymerichydrogel according to claim
 25. 52. The separation medium according toclaim 51 in the form of a membrane, slab, bead or column.
 53. Theseparation medium according to claim 51 being an electrophoretic mediumcapable of separating large biomolecules or compounds having a molecularweight of at least 2000 k.
 54. A method for separating one or morecompounds according to size using electrophoresis, the methodcomprising: (a) providing a medium in the form of polymeric hydrogelhaving a network containing macropores and micropores according to claim25; (b) adding one or more compounds to part of the medium; and (c)applying an electric potential causing at least one compound to passthrough the medium, wherein movement through the medium is related tothe size of the compound.
 55. A size exclusion electrophoresis systemcomprising: (a) a cathode; (b) an anode; and (c) a separation medium inthe form of polymeric hydrogel having a network containing macroporesand micropores according to claim 25 capable of separating a mixture ofcompounds according to size, the medium disposed between the anode andcathode.
 56. (canceled)